Ligands binding the complex of urokinase-type plasminogen activator (uppa) and its receptor (upar) that inhibit downstream upar interactions: identification and use in diagnosis or therapy

ABSTRACT

Antibodies or other ligands specific for the binary uPA-uPAR complexes, for ternary complexes comprising uPA-uPAR and for complexes of uPAR and proteins other than uPA such as integrins inhibit the interaction of uPA and uPAR with additional molecules with which the complexed interact. Such antibodies or other ligands are used in diagnostic and therapeutic methods, particularly against cancer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention in the field of biochemistry, immunology andmedicine relates to antibodies (“Abs”) or other ligands specific for (a)the binary uPA-uPAR complexes, (b) ternary complexes comprising uPA-uPARand (c) complexes of uPAR and proteins other than uPA such as integrins.These Abs or non-Ab ligands inhibit the interaction of uPA and uPAR withadditional molecules with which the above complexes interact. Such Absor other non-Ab ligands are used in diagnostic and therapeutic methods,particularly against cancer.

2. Description of the Background Art

A significant body of evidence from studies in vitro and in vivo hasestablished that the urokinase plasminogen activator (uPA) system iscentral to the process of metastasis, making it a promising target forcancer drug development (Mazar, A P et al. (1999) Angiogenesis 3:15-32). In addition to uPA, its cell surface receptor (uPAR) is asuitable target for the design and development of cancer therapeutic anddiagnostic agents (Mazar, A P (2001) Anti-Cancer Drugs 12: 397-400)because:

-   (a) uPAR is selectively expressed on metastatic tumor cells and    angiogenic endothelial cells (“ECs”), but not on other cells;-   (b) uPAR is an important participant in several extracellular and    intracellular pathways required for metastasis that are currently    the object of intense drug development efforts; and-   (c) it is possible to interfere at several different points along    the uPA pathway.    Thus, uPA and uPAR are promising targets for the development of    diagnostics and therapeutics useful against many different types of    tumors/cancers.

The uPA/uPAR System and Cancer

Metastasis and angiogenesis share many common functional features thatcharacterize invasive and migratory processes of tumor cells and of ECs.These features include (1) the up-regulation of protease and integrinexpression, (2) the loss of cell-cell and cell-matrix contacts, (3)increased responsiveness to growth and differentiation factors, and (4)remodeling of extracellular matrix (ECM) and basement membrane (BasM).All of these contribute to tumor progression.

The uPA “system,” which comprises the serine protease uPA, its receptoruPAR, and its specific serpin inhibitor, plasminogen activatorinhibitor-type 1 (PAI-1), plays a central role in many of theseactivities. The activity of this system is responsible for:

-   -   (1) initiating cascades that result in the activation of        plasminogen, activating several pro-metalloproteases (proMMPs),    -   (2) release and processing of latent growth factors such as        fibroblast growth factor-2 (FGF-2), vascular endothelial growth        factor (VEGF), hepatocyte growth factor (HGF), and transforming        growth factor-1 (TGFβ),    -   (3) (a) interactions with components of the ECM such as        vitronectin (Vn) and fibronectin (Fn),        -   (b) direct interactions with several integrins including            α5β1 and αvβ3, and (c) remodeling the BasM and ECM to            promote cell motility.            Further, the uPA system can also initiate localized fibrin            turnover which may play a role in angiogenesis.

The expression of uPA and uPAR has been demonstrated in numerous tumortypes including glioblastoma, prostate, breast, colon, hepatocellular,and renal cell carcinoma. (Mizukami I F et al. (1994) Clin Immunol andImmunopathol 71:96-104; Hsu D W et al., (1995) Am J Pathol 147:114-23;de Witte J H et al. (1999) Br J Cancer 79:1190-8). The expression of uPAand uPAR are typically greater in more aggressive forms of disease. Ontumor cells, this expression is often highest at the invasive front ofthe tumor. (Buo, L et al., (1995) Human Pathol 26:1133-1138; Yamamoto Met al. (1994) Cancer Res 54:5016-5020). Strong immunohistochemicalstaining for uPAR in blood vessels associated with the invasive front ofbreast, colon, and renal cell carcinomas has been reported (Bastholm Let al. Appl Immunohistochem Mol Morphol 7: 39-47; Nakata S et al. (1998)Int. J. Cancer 79:179-186). In the colon carcinoma study, uPARco-localized with VEGF. The expression of uPA and uPAR has also beenobserved on tumor-associated macrophages in several tumor types (OhtaniH et al. (1995) Int J Cancer 62:691-6; Xu Y et al. (1997) Hum Pathol28:206-13). uPA is chemotactic for monocytes and mediates both adhesionand migration of these cells. Adhesion and migration require only uPARoccupancy but not uPA catalytic activity. Thus, the uPA system isbelieved to contribute to tumor progression by acting on multipletumor-associated cell types.

Several recent studies have evaluated the therapeutic potential ofinhibiting the binding of uPA to uPAR in syngeneic systems. The deliveryof an adenovirus-encoded murine amino-terminal fragment of uPA(abbreviated “ATF”—this is the domain of uPA that contains the uPARbinding region) directly into tumors resulted in (a) suppression ofneovascularization and (b) arrest of tumor growth (Li H et al. (1998)Gene Ther 5:1105-1113). Due to species “specificity,” murine ATF wouldbe expected to bind only to murine host ECs and leukocytes, not to humantumor cells. This indicates that the tumor inhibition was mediatedthrough the suppression of the host angiogenic response. Finally, acollaborative study between some of the present inventors and S. Rabbaniand J. Gladu recently demonstrated that a polyclonal Ab raised against a100-residue fragment of rat uPAR selectively localized to a rat breasttumor which grew from cells of the Mat BIII cell line (Rabbani S A etal. (2002) Cancer Res 62:2390-97). This polyclonal antibody completelyinhibited tumor growth and led to tumor regression.

Unfortunately, despite the promise of targeting the uPA system fortherapeutic and diagnostic purposes, research efforts have not resultedin the development of agents suitable for the clinic. Small moleculeapproaches have been hampered by (1) the difficulty of potentlyinhibiting a protein-protein interaction (e.g., uPA-uPAR oruPAR-integrin), and (2) the lack of suitable leads or structuralinformation amenable to medicinal chemistry efforts. Several potentpeptide inhibitors of the uPA-uPAR interaction have been identified butthese would suffer from the typically poor pharmacological properties ofpeptides and have not demonstrated the requisite levels of activity evenin cell-based assays (Ploug M et al. (2001) Biochemistry 40:12157-68).

SUMMARY OF THE INVENTION

The present inventors produced a set of monoclonal antibodies (mabs)that bind to uPA-uPAR complexes and that inhibit their interaction ofwith downstream targets such as integrins. Such inhibition is expectedto inhibit tumor growth and metastasis. These mAbs may have utility as“naked” antibodies as well as for targeting therapeutic agents andimaging agents to tumors. Several antibodies that target uPAR areeffective in animal models of cancer growth (the A2780 ovarian cancermodel and the A549 lung cancer model). The epitopes recognized by thesemAbs are peptide regions within uPAR. Therefore uPAR peptidescorresponding to these regions or derived therefrom are useful asantagonists of uPAR interactions with downstream proteins.

It is common for malignant tumor cells and angiogenic ECs to gain aselective advantage in the process of cell migration and invasiveness.This advantage results at least in part from the cells' expression ofuPAR molecules on their surface, and these uPAR molecules are saturatedby binding the endogenously produced ligand, uPA.

Thus, mabs, peptides or other chemical entities that target andpreferably inhibit uPA-uPAR interactions with downstream targets areuseful in the treatment and/or diagnosis of cancer. Preferred downstreamligands of uPA-uPAR, or of uPAR alone, include integrins, low-densitylipoprotein receptor-related protein (LRP) as well as other bindingpartners. Some of these downstream ligands may mediate cell signaling,migration and/or invasion.

The present inventors have produced and studied two mabs, ATN-615 andATN-658, that specifically bind ligand-occupied uPAR and thus serve asexemplary molecules that can bind uPAR regardless of the presence ofligand. The mAbs can detect both occupied and unoccupied uPAR in a tumoror other diseased tissue where the uPA system plays a role in thepathobiology. Preferred Abs or other non-Ab ligands are those that donot bind to the uPA-binding site of uPAR.

The present inventors have identified the epitopes to which these Absbind. Such peptides or natural or synthetic peptides or peptidederivatives that retain the 3D structure of these epitopes are useful astherapeutic and/or diagnostic agents. Several peptide sequencesidentified based on these epitopes are disclosed herein.

In addition, the present inventors have developed a method to identifyAbs that mimic the characteristics of ATN-615 and ATN-658. This methodcan be used to develop humanized or fully human mAbs that recognize andbind to the same epitopes as those bound by ATN-615 and ATN-658. Suchmimics of ATN-615 and ATN-658, the latter of which has particularlyrobust anti-tumor activity, are included herein as therapeutic and/ordiagnostic agents.

The present invention is further directed to macromolecules, includingAbs, antigen binding fragments such as single chain Abs=(scFv), non-Abpolypeptides and peptides, aptamers, etc., as well as small organicmolecules, that have the property of binding to uPAR without inhibitingthe binding of uPA. Some of these molecules interfere with downstreaminteractions of either uPA-uPAR or uPAR alone.

In addition to specific compositions that target uPA-uPAR interactions,this invention is also directed to methods for detecting Abs that bindexclusively to uPA-uPAR or that inhibit downstream interactions of uPAR.Thus, the invention includes a method for identifying these uPA-uPARcomplex-binding molecules. This method may be varied to detect moleculesthat bind other components or complexes of the uPA/uPAR system. Forexample, uPA bound to its natural inhibitor PAI-1 also binds uPAR,forming a uPA:PAI-1/uPAR ternary complex. One method of the presentinvention comprises using such ternary complexes to screen for ligandswhich interact only with this complex but not with the binary uPA:PAI oruPA-uPAR complex. Another method is directed to inhibitors thatinterfere with the interaction of the ternary complexes with down-streamtargets such as LRP.

Such an approach is suitable to identify ligand molecules that becomeinternalized when bound to the complex.

In addition, this invention includes methods to detect a molecule thatbinds to the uPA-uPAR complex (but not to uncomplexed uPA or uPAR) or todetect inhibitors that interfere with the binding of uPA-uPAR or uPAR todownstream targets as well as the binding ligands themselves. Suchbinders may be Abs, others proteins, peptides, aptamers, smallmolecules, etc. A specific embodiment of this type would be a uPA-uPARor uPAR ligand that interfered with uPAR mediated assembly of Fn or thatperturbed the binding of Fn or Fn fragments to the integrin α₅β₁.Alterations of the assembly of other matrix components (e.g.,vitronectin) are also covered by this invention.

This invention is also directed to methods for identifying inhibitors ofplasminogen activation that do not inhibit uPA catalytic activity andnovel compositions that have this activity.

More specifically, the present invention is directed to a ligand thatbinds to a binary uPA-uPAR complex, which ligand does not substantiallybind to (a) free uPA or (b) the region of uPAR that recognizes and bindsto uPA, so that the ligand does not inhibit uPA-uPAR binding.

Another embodiment comprises a ligand that binds to a ternary complex ofuPA-uPAR and an additional molecule (X), such as PAI-1, which ligand:

-   (a) binds to a uPA-uPAR-X complex,-   (b) does not substantially bind to any of the following: (i) a    uPA-uPAR complex, (ii) a uPA-X complex, (iii), the uPA-recognizing    and uPA-binding region of uPAR or of X, (iv) free uPA, or (v), free    X; and-   (c) does not substantially inhibit uPA-uPAR binding or uPA-X    binding.    Preferably, the above ligand substantially does not bind to free    uPAR.

The above ligand may be a polypeptide, preferably an Ab, such as a mAb,or an antigen binding fragment thereof. Preferred mAbs are humanizedchimeric or human mAbs.

In one embodiment, a preferred mAb or antigen-binding fragmentcomprises:

-   (a) a V_(L) chain comprising three CDR's which have the respective    amino acids sequences SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and-   (b) a V_(H) chain comprising three CDR's which have the respective    amino acids sequences SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.    A more preferred mAb or antigen binding fragment, as above,    comprises-   (a) a V_(L) chain with the sequence SEQ ID NO: 1; and-   (b) a V_(H) chain with the sequence SEQ ID NO:2.

In another preferred embodiment, the mAb or antigen-binding fragmentcomprises:

-   (a) a V_(L) chain comprising three CDR's which have the respective    amino acids sequences SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13;    and-   (b) a V_(H) chain comprising three CDR's which have the respective    amino acids sequences SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.    A more preferred mAb or antigen binding fragment, as above,    comprises:-   (a) a V_(L) chain has the sequence SEQ ID NO:9; and-   (b) a V_(H) chain has the sequence SEQ ID NO:10.

A preferred Ab of the present invention is one selected from: (a) mAbdesignated ATN-615 produced by hybridoma having ATCC Accession #_); (b)a mAb designated ATN-658 produced by a hybridoma having ATCC Accession#_; (c) a mAb having essentially the same antigen-bindingcharacteristics as ATN-615; and (d) a mAb having essentially the sameantigen-binding characteristics as ATN-658.

In one embodiment, the above ligand is one that inhibits binding ofuPA-uPAR complexes with another biological ligand for these complexes.Examples of “other biological ligands” include integrins, preferablyα5β1, αvβ3, αvβ5, α3β1, α6β1, or α4β1.

The above ligand may be one that ligand interferes with and inhibits (a)uPAR mediated assembly of Fn, (b) binding of Fn or a fragment thereof tointegrin α₅β₁, or (c) the assembly of Vn components.

In a preferred embodiment, the above ligand is (a) diagnosticallylabeled (with a detectable label); or (b) labeled with, conjugated to,or fused to (in the case of a polypeptide), a therapeutically activemoiety, rendering the ligand therapeutically active.

Provided herein is a diagnostic composition comprising (a) thediagnostically labeled ligand as above; and (b) a diagnosticallyacceptable carrier.

In the diagnostic composition the ligand is preferably labeled with aradionuclide, a PET-imagable agent, an MRI-imagable agent, a fluorescer,a fluorogen, a chromophore, a chromogen, a phosphorescer, achemiluminescer or a bioluminescer. Preferred radionuclides are selectedfrom the group consisting of ³H, ¹⁴C, ³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁷Ru,⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I, ¹⁶⁹Yb and ²⁰¹Tl. Preferred fluorescers orfluorogens are fluorescein, rhodamine, dansyl, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde, fluorescamine, afluorescein derivative, Oregon Green, Rhodamine Green, Rhodol Green andTexas Red.

The present invention provides a therapeutic anti-angiogenic oranti-tumor pharmaceutical composition that inhibits undesiredangiogenesis, tumor growth and/or tumor metastasis comprising (a) aneffective amount of the therapeutically active ligand above, and (b) apharmaceutically acceptable carrier. This composition is preferably in aform suitable for injection. The therapeutically active moiety may beconjugated directly to, or bound indirectly to, the ligand. A preferredtherapeutic moiety is a chemotherapeutic drug, a toxin or a therapeuticradionuclide (preferably ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²⁵I, ¹³¹I, ¹⁸⁶Re,¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb or ²¹⁷Bi).

In the above therapeutic composition, the therapeutically active moietymay be a peptide or polypeptide, e.g., a toxin, which is fused to theligand.

This invention is directed to a method for inhibiting cell migration,cell invasion, cell proliferation or angiogenesis, or for inducingapoptosis, comprising contacting cells associated with undesired cellmigration, invasion, proliferation or angiogenesis with an effectiveamount of the above therapeutically active ligand

Also included is a method for treating a subject having a disease,disorder or condition characterized by undesired angiogenesis, tumorgrowth and/or tumor metastasis comprising administering to the subjectan effective amount of the above therapeutic pharmaceutical composition.

Also provided is an assay method for detecting in a sample a substancesuspected of having the binding properties of the above ligand,comprising

(a) contacting the sample with uPA-uPAR complexes and determiningbinding of a component of the sample to the complexes;

(b) contacting the sample with free uPAR and determining binding of acomponent of the sample to the uPAR.

(c) comparing the binding of (a) and (b),

wherein the presence of binding in (a) and a substantial absence orsignificantly lower binding in (b) is indicative of the present of thesubstance in the sample.

The assay may be a competitive binding assay using a labeled bindingpartner that binds to uPA-uPAR complexes, wherein the substance in thesample competes for binding with the binding partner.

One embodiment is an assay method for detecting in a sample a substancesuspected of having the binding properties of the above ligand,comprising

(a) contacting the sample with uPA-uPAR-X complexes (with X defined asabove) and determining binding of a component of the sample to thecomplexes;

(b) contacting the sample with one or more of (i) uPA:X complexes; (ii)uPA-uPAR complexes; or (iii) uncomplexed X, and determining binding of acomponent of the sample to uPA-X, uPA-uPAR or X;

(c) comparing the binding of (a) and (b),

wherein the presence of binding in (a) and a substantial absence orsignificantly lower binding in (b) is indicative of the present of thesubstance in the sample.

In the above method, the complexes may be on a cell surface

This invention includes an isolated peptide comprising at least 3 aminoacids, which peptide, when part of a longer amino acid sequence, ispresent in a linear epitope bound by a mAb which has (a) a V_(L) chaincomprising three CDR's which have the respective amino acids sequencesSEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; and (b) a V_(H) chaincomprising three CDR's which have the respective amino acids sequencesSEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A preferred isolated peptideas above is present in a linear epitope bound by a mAb with (A) a V_(L)chain that has the sequence SEQ ID NO:1; and (b) a V_(H) chain that hasthe sequence SEQ ID NO:2.

In another embodiment, the isolated peptide comprises at least 3 aminoacids, and peptide, when it is part of a longer amino acid sequence, itis present in a linear epitope bound by a mAb having (a) a V_(L) chaincomprising three CDR's which have the respective amino acids sequencesSEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; and (b) a V_(L) chaincomprising three CDR's which have the respective amino acids sequencesSEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16. A preferred isolatedpeptide as above is present in a linear epitope bound by a mAb having(a) a V_(L) chain that has the sequence SEQ ID NO:9; and (b) a V_(H)chain that has the sequence SEQ ID NO:10.

The invention is also directed to an isolated peptide, or a substitutionvariant thereof, comprising at least 3 amino acids, which peptide, whenpart of a longer amino acid sequence, is present in a linear epitoperecognized by the mAb designated ATN-615 or by the mAb designatedATN-658.

The invention includes an assay method for identifying an Ab or otherligand that binds to the same epitope as does mAb ATN-615 or mAb ATN-658comprising measuring the ability of a sample suspected of containing theAb or other ligand to competitively inhibit the binding of detectablylabeled ATN-615 or ATN-658 to (i) immobilized suPAR, (ii) immobilizedsuPAR D2D3 or (iii) an immobilized fragment of suPAR or D2D3 of suPAR,wherein competitive inhibition of at least about 20%, preferably 50%,more preferably 70% and most preferably 90%, indicates that an antibodyor ligand binds to the same epitope.

One embodiment is a method for identifying a peptide that is recognizedby (a) ATN-615, (b) ATN-658, or (c) an Ab or other ligand that with thesame binding specificity as ATN-615 or ATN-658, which method comprisesmeasuring the ability of a sample suspected of containing the peptide,or a candidate peptide, to competitively inhibit the binding ofdetectably labeled ATN-615 or ATN-658 or the Ab or other ligand with thesame binding specificity, to (i) immobilized suPAR, (ii) immobilizedsuPAR D2D3 or (iii) an immobilized fragment of suPAR or D2D3 of suPAR,wherein competitive inhibition of at least about 20%, preferably 50%,more preferably 70% and most preferably 90%, indicates that the peptidehas the binding specificity.

Included herein is an assay to screen for a compound, or to determinewhether a candidate compound has essentially the same bindingcharacteristics to a uPAR structure as does ATN-615 or ATN-658,comprising measuring the ability of a sample being screened or thecandidate compound to competitively inhibit the binding of detectablylabeled ATN-615 or ATN-658 to (i) immobilized suPAR, (ii) immobilizedsuPAR D2D3 or (iii) an immobilized fragment of suPAR or D2D3 of suPAR,wherein competitive inhibition of at least about 20%, preferably 50%,more preferably 70% and most preferably 90%, indicates that the peptidehas the binding characteristics.

In one embodiment of the foregoing assay, the compound being screenedfor, or the candidate compound, is a small organic molecule having amolecular mass between about 50 Da and about 2500 Da. In anotherembodiment, the compound being screened for, or the candidate compound,is a nucleic acid molecule, preferably an oligonucleotide such as anRNAi molecule or an aptamer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. SDS-PAGE analysis of ATF and suPAR fragments expressed in S2cells. ATF (aa 1-143) and suPAR (aa 1-279) were cloned and expressed inDrosophila Schneider S2 cells. Cells were induced to express recombinantproteins with copper (0.5 nM) for 7 days. Culture supernatants werecollected and clarified by centrifugation and filtration. After additionof protease inhibitors proteins were purified by ion exchangechromatography on either DEAE-Sepharose, pH 7.5, (ATF) or SP-Sepharose,pH 8.8, (suPAR). ATF and suPAR were further purified using RP-HPLC.Purified, recombinant suPAR was digested with chymotrypsin to generatethe soluble domain2/domain3 (D2D3) fragment. Prior to immunization, D2D3protein was conjugated to the carrier protein keyhole limpet hemocyanin(KLH).

FIG. 2. ATN-658 binds to a non-glycosylated mutant of suPAR, indicatingthat ATN-658 is directed against a peptide (not a carbohydrate) epitope,like most other anti-uPAR mabs.

FIG. 3. Western blots with two anti-D2D3 mabs, ATN-615 and ATN-658.Recombinant proteins were resolved by SDS-PAGE and transferred to PVDFmembranes. Membranes were probed with purified antibodies (5 μg/ml).ATN-615 and ATN-658 specifically recognize suPAR and D2D3.

FIG. 4. Anti-D2D3 antibodies inhibit uPA-induced migration. Migration ofuPAR expressing CHO cells toward uPA (500 nM) was determined using amodified Boyden chamber assay. Anti-integrin α5, anti-uPAR, andanti-D2D3 antibodies inhibit migration, suggesting that integrin α5β1and uPAR are critical for uPA-induced migration.

FIG. 5. ¹²⁵I-labeled ATN-658 binds to HeLa cells with high affinity.Confluent monolayers of HeLa cells in 24-well plates were incubated withincreasing concentrations of [¹²⁵I]-ATN-658 at room temperature for onehour. Cells were washed extensively with PBS/Tween-20 and bound materialwas solubilized with 1 M NaOH. Non-specific binding was determined inthe presence of a 100-fold excess of unlabeled Ab.

FIG. 6. shows that the mAb ATN-658 does not compete with binding of uPAto HeLa cells. Binding of ATN-658 to HeLa cells did not inhibit bindingof ¹²⁵I-scuPA. HeLa cells were incubated with 5 nM ¹²⁵I-scuPA in thepresence or absence of either 300 nM unlabeled scuPA or 300 nM ATN-658.ATN-617, an anti-uPAR mAb that blocks the binding of uPA to uPAR isshown to compete with scuPA binding.

FIG. 7 shows that ATN-658 inhibits tumor growth in the A2780 ovariancancer model as effectively as cisplatin. A2780 cells express only uPARand not uPA.

FIG. 8 shows that ATN-658 inhibits tumor growth in an A549 lung cancer(non-small cell) model in which 10⁶ tumor cells were inoculated. A549cells express both uPA and uPAR.

FIG. 9 shows that biotinylated ATN-658 binds saturably to suPAR.

FIGS. 10A and 10B both show results of a competition assay usingbiotin-labeled ATN-658 to identify mAbs that recognize the same epitopeon suPAR. ATN-616 and ATN-617 are anti-uPAR antibodies that block thebinding of uPA to uPAR. ATN-616 specifically binds ligand-occupied uPAR.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventors have found that mabs, peptides or other chemicalentities that target the uPA/uPAR complex or the uPAR-integrin complexare useful in the treatment and/or diagnosis of cancer. To date, thepresent inventors believe that no antibodies have been described thatrecognize the uPA-uPAR complex but not (a) uPAR or uPA individually or(b) uPAR in the presence of uPA (i.e., ligand occupied uPAR).

Further, the uPA-uPAR complex or uPAR alone have other “downstream”ligands such as integrins, low-density lipoprotein receptor-relatedprotein (LRP) and other binding partners. These downstream interactionsare believed to be important to the processes of cell migration,invasion and proliferation. It is thus desirable processes to targetthese processes therapeutically or detect the process or theirinteracting components diagnostically.

In addition to specific antibodies that target these interactions, asdescribed in more detail below, this invention is also directed tomethods for detecting antibodies that bind exclusively to the uPA-uPARcomplex or that inhibit downstream interactions of uPAR.

The Antibody Approach

The present inventors have generated a panel of mAbs targeting uPAR.uPAR is an ideal target for antibodies because it is expressed on thecell surface. Expression of uPAR at the tumor-vasculature interface (oninvasive tumor cells, angiogenic endothelial cells, or tumor-associatedmacrophages) suggests that antibodies targeting this protein would notsuffer the same barriers to diffusion that have led to the failure ofother mAbs to enter tumors and serve as diagnostic agents or exerttherapeutic effects. Importantly, uPAR is not normally expressed onquiescent tissues, which should minimize the potential for toxicity whenemploying a therapeutic Ab and minimize non-specific signals (or falsepositives) when employing a diagnostic Ab.

The present inventors have raised mAbs against a fragment of the solubleform of uPAR (known as “suPAR”) expressed in Drosophila S2 cells. Insuch cells, a minimally glycosylated isotype of suPAR is expressed. Useof this suPAR as an immunogen is expected to overcome the heterogeneousbinding to uPAR observed with all other mAbs examined to date. Studiesperformed as part of a Leukocyte Antigen Workshop compared anti-uPARantibodies available in 1995-1996 and found all of them to be specificfor carbohydrate, not protein, epitopes (Manupello, J. et al., (1996)Tiss. Antigens 48: 368). Indeed, uPAR expressed in tumors is highly andheterogeneously glycosylated, and the glycosylation pattern andrepresentation of different isoforms change in response to varioussignals (Stoppelli M P et al. (1985) Proc. Natl. Acad. Sci. USA 824939-4943). Thus, anti-uPAR antibodies raised against carbohydrateepitopes are unlikely to recognize all isoforms of uPAR and maycross-react undesirably with other proteins expressing glycosylationstructures similar to those present on uPAR. Use of S2 has led to theidentification of mAbs that recognize the protein epitopes within suPAR(FIG. 2).

The present inventors have produced stable clones that express highamounts of suPAR as well as domain fragments of suPAR. Typical yieldsusing these expression systems are on the order of 25-50 mgs/L afterpurification (>95% pure). Thus, the present inventors have shown that itis possible to express all the components required for the generation ofthe antibodies of the present invention and to design assays to evaluateand characterize them.

A mutant form of suPAR has been expressed in which all glycosylationsites have been mutated. The existing murine mAb clones may be humanizedor primatized.

The present inventors' ability to generate conformationally intactdomain fragments of suPAR has allowed them to produce mAbs againstisolated D1 and isolated D2D3 (of suPAR). An epitope exposed in the uPARD2D3 fragment is also exposed in full length, intact uPAR only afterbinding of uPA. This epitope has been demonstrated to be critical to thepro-migratory activity of uPA (Andolfo A et al. (2002) Thromb Haemost88:298-306). Thus, antibodies generated against the D2D3 fragment wherethis epitope is already exposed, are expected to have anti-migratoryactivity. This has been demonstrated for mAbATN-658.

This invention is thus directed in part to a mAb that binds to a binaryuPA-uPAR complex, but not substantially to (a) free uPA or (b) theregion of uPAR that recognizes and binds to uPA, so that the mAb doesnot inhibit uPA-uPAR binding, which mAb is produced by a processcomprising the initial step of immunizing a mammal, preferably a mouse,with

-   (a) a minimally glycosylated isotype of suPAR expressed in    Drosophila cells, or-   (b) a de-glycosylated mutant of suPAR in which 4 or 5 glycosylation    sites have been mutated.    Following immunization using standard protocols, conventional    techniques are employed to generate hybridoma cell lines from the    immunized animals and to generate mAbs having the desired    properties. mAbs specific for uPA-uPAR complexes having additional    or somewhat different properties as disclosed herein are made in the    same way using the same novel suPAR antigens. The mAbs made by this    process may or may not bind free uPAR in solution.

There are five N-linked glycosylation sites in wild-type uPAR: Asn⁵² (inD1) Asn¹⁶² and Asn¹⁷² (in D2) and Asn²⁰⁰ and Asn²³³ (in D3). The latterfour sites in D2 and D3 are preferably mutated to Gln to generate apreferred de-glycosylated suPAR immunogen for raising mAbs of theinvention.

Anti-D2D3 mAbs have also been generated which recognize uPAR on cellsurfaces regardless of whether the uPAR is occupied by uPA. Since alarge percentage of uPAR on tumors indeed is bound to uPA, antibodies ofthis specificity are useful as targeting agents for therapeutic anddiagnostic moieties. In addition, in cancer patients, it is frequentlyobserved that tumors express uPAR that is cleaved by proteases expressedby these same tumors, leaving a residual D2D3 fragment still attached tothe tumor (Sier C F et al., Thromb Haemost. 2004, 91:403-11). Thus,successful targeting of these tumors require anti D2D3 antibodies. Theseantibodies are also useful for in vivo imaging applications.

The anti-D2D3 antibodies (and other antibodies of the present invention)are tested preferably in xenogeneic tumor models, two preferred examplesof which are the A2780 and A549 models (described in more detail below).

Variable (V) Region Amino Acid Sequences of Two Preferred mAbs

mAb ATN-658: Variable Region Sequences

The consensus amino acid sequence (single-letter code) of the lightchain variable region (V_(L)) and heavy chain variable region (V_(H))polypeptides of mAb ATN-658 are shown below. cDNA was prepared fromtotal RNA extracted from the hybridoma expressing ATN-658 and thevariable regions were cloned, amplified and sequenced using standardtechniques. The complementarity-determining regions (CDRs) for eachvariable region are highlighted (italic, bold, underscored)

ATN-658 V_(L) Consensus Protein (SEQ ID NO:1):

  1 DIXLTQSPLT LSVTIGQPAS ISC

 

W LLQRPGQSPK  51 RLIY

 

GVPDRFTGS GSGTDFTLKI SRVEAEDLGV YYC

101 LTFGAGTKLE LKL

ATN-658 V_(H) Consensus Protein (SEQ ID NO:2)

  1 VQLQESGPEL VKTGASVKIS CKAS

 

WVKQSH GKSLEWIG

 51

 

RATFT VDTSSRTAYM QFNSLTSEDS AVYYCAR

101

WGQ GTTVTVS

TABLE 1 Characteristics of CDRs of ATN-658 L and H Chains No. of SEQCDR* residues Sequence¹ ID NO: CDR L1 16 KSSQSLLDSDGKTYLN 3 CDR L2 7LVSKLDS 4 CDR L3 9 WQGTHFPLT 5 CDR H1 10 GYSFTSYYMH 6 CDR H2 17EINPYNGGASYNQKIKG 7 CDR H3 10 SIYGHSVLDY 8 *CDR-L1: first GDR of Lchain; CDR-H2: 2^(nd) CDR of H chain, etc.

mAb ATN-615: Variable Region Sequences

Amino acid sequence (single-letter code) of the light chain (V_(L)) andheavy chain (V_(H)) variable regions of monoclonal antibody ATN-615.cDNA was prepared from total RNA extracted from the hybridoma expressingATN-615 and the variable regions cloned, amplified and sequenced usingstandard techniques. The complementarity-determining regions (CDRs) foreach variable region are highlighted in red.

ATN-615 V_(L) Consensus Protein Sequence (SEQ ID NO:9)

  1 DIVLTQSPDI TAASLGQKVT ITC

 

WYQQKSG TSPKPWIF

 51

GVPAR FSGSGSGTSY SLTISSMEAE DAAIYYC

 

FGGGT 101 KLEIKR

ATN-615 V_(H) Consensus Protein Sequence (SEQ ID NO:10)

  1 VKLQQSGPEV VKPGASVKIS CKAS

 

WVKQRP GQGLEWIG

 51

 

KATLT ADTSSSTAYM QLSSLTSEDS AVYFCAR

101

WGQG TTVTVSS

TABLE 2 Characteristics of the CDRs of ATN-615 No. of SEQ ID CDR*residues Sequence NO: CDRL1 10 SASSSVSYMH 11 CDRL2 7 EISKLAS 12 CDRL3 8QQWNYPFT 13 CDRH1 10 GYSFTNFYIH 14 CDRH2 17 WIFHGSDNTEYNEKFKD 15 CDRH3 9WGPHWYFDV 16 *CDR-L1: first CDR of L chain; CDR-H2: 2^(nd) CDR of Hchain, etc.

According to the present invention, an Ab or mAb, has “essentially thesame antigen-binding characteristics” as a reference mAb if itdemonstrates a similar specificity profile (e.g., by rank ordercomparison), and has affinity for the relevant antigen (e.g., uPA-uPARcomplex) within 1.5 orders of magnitude, more preferably within oneorder of magnitude, of the reference Ab.

The antibodies are evaluated for direct anti-angiogenic activity in anin vivo Matrigel plug model. Radioiodinated antibodies are used to testAb internalization using in MDA MB 231 cells which express both receptorand ligand. Antibody internalization is also measured in the presence ofPAI-1:uPA complexes.

Antibodies Specific for uPA, uPAR and Binary and Ternary Complexesthereof

In the following description, reference will be made to variousmethodologies known to those of skill in the art of immunology, cellbiology, and molecular biology. Publications and other materials settingforth such known methodologies to which reference is made areincorporated herein by reference in their entireties as though set forthin full. Standard reference works setting forth the general principlesof immunology include Abbas, A K et al., Cellular and MolecularImmunology (Fourth Ed.), W.B. Saunders Co., Philadelphia, 2000; Janeway,C A et al., Immunobiology. The Immune System in Health and Disease, 4thed., Garland Publishing Co., New York, 1999; Roitt, I et al.,Immunology, (current ed.) C.V. Mosby Co., St. Louis, Mo. (1999); Klein,J, Immunology, Blackwell Scientific Publications, Inc., Cambridge,Mass., (1990).

Monoclonal antibodies (mAbs) and methods for their production and useare described in Kohler and Milstein, Nature 256:495-497 (1975); U.S.Pat. No. 4,376,110; Hartlow, E. et al., Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988);Monoclonal Antibodies and Hybridomas: A New Dimension in BiologicalAnalyses, Plenum Press, New York, N.Y. (1980); H. Zola et al., inMonoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press,1982)).

Immunoassay methods are also described in Coligan, J E et al., eds.,Current Protocols in Immunology, Wiley-Interscience, New York 1991 (orcurrent edition); Butt, W R (ed.) Practical Immunoassay: The State ofthe Art, Dekker, New York, 1984; Bizollon, C A, ed., MonoclonalAntibodies and New Trends in Immunoassays, Elsevier, New York, 1984;Butler, J E, ELISA (Chapter 29), In: van Oss, C J et al., (eds),IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803;Butler, J E (ed.), Immunochemistry of Solid-Phase Immunoassay, CRCPress, Boca Raton, 1991; Weintraub, B, Principles of Radioimmunoassays,The Endocrine Society, March, 1986; Work, T S et al., LaboratoryTechniques and Biochemistry in Molecular Biology, North HollandPublishing Company, NY, 1978; Dabbs, D J, DiagnosticImmunohistochemistry, Churchill Livingstone, 2001.

Anti-idiotypic antibodies are described, for example, in Idiotypy inBiology and Medicine, Academic Press, New York, 1984; ImmunologicalReviews Vol. 79, 1984 and Vol. 90, 1986; Curr. Top. Microbiol., Immunol.Vol. 119, 1985; Bona, C. et al., CRC Crit. Rev. Immunol., pp. 33-81(1981); Jerne, N K, Ann. Immunol. 125C:373-389 (1974); Urbain, J et al.,Ann. Immunol. 133D:179- (1982); Rajewsky, K. et al., Ann. Rev. Immunol.1:569-607 (1983).

The present invention provides antibodies, both polyclonal andmonoclonal, reactive with uPA/uPAR complexes that inhibit interactionsof uPAR with integrins or other downstream targets. The antibodies maybe xenogeneic, allogeneic, syngeneic, or modified forms thereof, such ashumanized or chimeric antibodies. Antiidiotypic antibodies specific forthe idiotype of, for example, an anti-uPA/uPAR Ab are also included. Theterm “antibody” is also meant to include both intact molecules as wellas fragments thereof that include the antigen-binding site and arecapable of binding to a target epitope of, e.g., uPA/uPAR oruPAR-integrin complex. These include, Fab and F(ab′)₂ fragments whichlack the Fc fragment of an intact Ab, clear more rapidly from thecirculation, and may have less non-specific tissue binding than anintact Ab (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Also includedare Fv fragments (Hochman, J. et al. (1973) Biochemistry 12:1130-1135;Sharon, J. et al. (1976) Biochemistry 15:1591-1594)). These variousfragments are produced using conventional techniques such as proteasecleavage or chemical cleavage (see, e.g., Rousseaux et al., Meth.Enzymol., 121:663-69 (1986))

Polyclonal antibodies are obtained as sera from immunized animals suchas rabbits, goats, rodents, etc. and may be used directly withoutfurther treatment or may be subjected to conventional enrichment orpurification methods such as ammonium sulfate precipitation, ionexchange chromatography, and affinity chromatography (see Zola et al.,supra).

An immunogen for generation of the antibodies of this invention maycomprise uPAR, suPAR, uPA/uPAR or uPAR-integrin complexes/or anepitope-bearing fragments or derivative thereof. Useful immunogens areproduced in a variety of ways known in the art, e.g., expression ofcloned genes using conventional recombinant methods, isolation fromcells of origin, cell populations expressing high levels of e.g., uPA oruPAR, etc. In the case of shorter fragments, they may be chemicallysynthesized. A preferred immunogen is the D2D3 fragment of suPAR.

The mAbs may be produced using conventional hybridoma technology, suchas the procedures introduced by Kohler and Milstein (Nature, 256:495-97(1975)),—and modifications thereof (see above references). An animal,preferably a mouse is primed by immunization with an immunogen as aboveto elicit the desired Ab response in the primed animal.

B lymphocytes from the lymph nodes, spleens or peripheral blood of aprimed, animal are fused with myeloma cells, generally in the presenceof a fusion promoting agent such as polyethylene glycol (PEG). Any of anumber of murine myeloma cell lines are available for such use: theP3-NS1/1-Ag4-1, P3-x63-k0Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma lines(available from the ATCC, Rockville, Md.). Subsequent steps includegrowth in selective medium so that unfused parental myeloma cells anddonor lymphocyte cells eventually die while only the hybridoma cellssurvive. These are cloned and grown and their supernatants screened forthe presence of Ab of the desired specificity, e.g., by immunoassaytechniques. Positive clones are subcloned, e.g., by limiting dilution,and the mAbs are isolated.

Hybridomas produced according to these methods can be propagated invitro or in vivo (in ascites fluid) using techniques known in the art(see generally Fink et al., Prog. Clin. Pathol., 9:121-33 (1984)).Generally, the individual cell line is propagated in culture and theculture medium containing high concentrations of a single mAb can beharvested by decantation, filtration, or centrifugation.

Production of mAbs

A preferred approach for producing a mAb according to the presentinvention is as follows. D2D3 is prepared from suPAR using chymotrypticdigest and purification (Shliom, O. et al., (2000) J. Biol. Chem.275:24304-12). D2D3 is then conjugated to any useful carrier proteinsuch as albumin, keyhole limpet hemocyanin (KLH) or ovalbumin.Immunizations are typically carried out in complete Freund's adjuvantfollowed by periodic boosts in incomplete Freund's adjuvant. Animals arealso bled periodically and the titer of the serum measured using anELISA in which suPAR is immobilized to the surface of microplate wells.

If a peptide is used, it is preferably conjugated to a carrier protein,e.g., KLH, is and injected into BALB/c mice intraperitoneally (i.p.) incomplete Freund's adjuvant (e.g., 50 μg conjugate), followed by twoadditional injections of the same dose in incomplete Freund's adjuvantat two week intervals. After one month, a final injection is given i.p(e.g., 50 μg in 0.5 ml PBS) and preferably also intravenously (i.v.)(e.g., 50 μg in 0.2 ml) without adjuvant.

Spleen cells are harvested three days after the final injection andfused with P3X63AF8/653 or other myeloma cells using standardtechniques.

Test Cells for Screening and Characterizing Antibodies

Pure suPAR immobilized onto plastic is preferred for the primaryscreening. Cells such as the HeLa line that overexpress uPAR may also beused to demonstrate cell binding of an anti-suPAR mAb. Many tumor celllines overexpressing uPAR are well-known and publicly available; thesemay be used for screening. Cells are generally plated in 96-wellmicroplates. The cells may be fixed, e.g., with methanol/acetone(50/50), and the binding detected by immunofluorescence staining.Alternatively, the mAbs may be radiolabeled and binding detected bymeasurement of radioactivity.

In one embodiment, a hybridoma supernatant (e.g., 50 μl) is added towells containing fixed 293 cells for about 1.5 h at 37° C. Plates arewashed twice in washing buffer (such as PBS/0.05% Tween-20), andRhodamine Red-conjugated goat anti-mouse IgG is added (e.g., 30 μl/well)at an appropriate dilution, such as 1:100, for 1.5 h at 37° C. Afterwashing in a washing buffer, cells are examined for the presence ofimmunofluorescence; in the embodiment described here, fluorescencemicroscopy is used.

In this embodiment, immunofluorescence is the basis for determiningwhether a hybridoma supernatant contains an Ab specific for the uPA/uPARcomplex (although immunohistochemical staining may also be used). Ifsupernatants show positively staining the hybridoma clones are selected,expanded and the supernatants tested for reactivity to the complex byELISA.

In a preferred ELISA, the peptide is coupled to ovalbumin (OVA) as acarrier protein and the peptide/OVA conjugate coated onto wells of 96well EIA plate which receives, for example, 2 μg/ml of conjugate in 50μl coating buffer (0.2 M Na₂CO₃/NaHCO₃, pH9.6). Plates are incubatedovernight at 4° C., blocked with an appropriate blocking buffer, e.g.,PBS containing 1% BSA (200 μl/well) overnight at 4° C. Hybridomasupernatants (e.g., 50 μl) are added to wells for 1.5 hours at roomtemperature. Plates are washed twice in washing buffer (e.g., PBS/0.05%Tween-20), and enzyme-coupled secondary Ab, such as alkalinephosphatase-coupled goat-anti-mouse IgG is added (50 μl/well) at anappropriate dilution, e.g., 1:2000. Plates are incubated for 1.5 hoursat RT. After washing 4× in washing buffer, an appropriate chromogenicsubstrate for the enzyme, e.g., CP-nitrophenylphosphate in thisembodiment (available from Kirkegaard and Perry Co., Gaithersburg, Md.),is added for about 30 min and absorbance measured at wavelengthappropriate for the colored product (here 405 nm). Hybridomasupernatants that react strong with the epitope-bearing peptide (e.g.,A₄₀₅>1.0 when negative controls are <0.02) are re-cloned (preferablytwice), and the mab reactivity again confirmed by ELISA as above.

The term “antibody” is meant to include both intact immunoglobulin (Ig)molecules as well as fragments and derivative thereof, that may beproduced by proteolytic cleavage of Ig molecules or engineeredgenetically or chemically. Fragments include, for example, Fab, Fab′,F(ab′)₂ and Fv, each of which is capable of binding antigen. Thesefragments lack the Fc fragment of intact Ab and have an additionaladvantage, if used therapeutically, of clearing more rapidly from thecirculation and undergoing less non-specific tissue binding than intactantibodies. Papain treatment of Ig's produces Fab fragments; pepsintreatment produces F(ab′)₂ fragments. These fragments may also producedby genetic or protein engineering using methods well known in the art. AFab fragment is a multimeric protein consisting of the portion of an Igmolecule containing the immunologically active portions of an Ig heavy(H) chain and an Ig light (L) chain covalently coupled together andcapable of specifically combining with antigen. Fab fragments aretypically prepared by proteolytic digestion of substantially intact Igmolecules with papain using methods that are well known in the art.However, a Fab fragment may also be prepared by expressing in a suitablehost cell the desired portions of Ig H chain and L chain using methodswell known in the art. A (Fab′)₂ fragment is a tetramer that includes afragment of two H and two L chains. The Fv fragment is a multimericprotein consisting of the immunologically active portions of an Ig Hchain variable (V) region (V_(H)) and an Ig L chain V region (V_(L))covalently coupled together and capable of specifically combining withantigen. Fv fragments are typically prepared by expressing in suitablehost cell the desired portions of Ig V_(H) region and V_(L) region usingmethods well known in the art.

Single-chain antigen-binding protein or single chain Ab, also referredto as “scFv,” is a polypeptide composed of an Ig V_(L) amino acidsequence tethered to an Ig V_(H) amino acid sequence by a peptide thatlinks the C-terminus of the V_(L) sequence to the N-terminus of theV_(H) sequence.

In a preferred embodiment, the Ab is a mAb designated ATN-615 orATN-658, both of which are IgG1 antibodies.

In another preferred embodiment, the Ab is a chimeric Ab that recognizesan epitope recognized by ATN-615 or ATN-658.

Chimeric Antibodies

The chimeric antibodies of the invention comprise individual chimeric Hand L Ig chains. The chimeric H chain comprises an antigen bindingregion derived from the H chain of a non-human Ab specific for e.g.,uPA/uPAR or uPAR-integrin complex, for example, mAb ATN-615 or ATN-658,which is linked to at least a portion of a human C_(H) region. Achimeric L chain comprises an antigen binding region derived from the Lchain of a non-human Ab specific for the target antigen, such as thehybridoma ATN-615 or ATN-658, linked to at least a portion of a humanC_(L) region. As used herein, the term “antigen binding region” refersto that portion of an Ab molecule which contains the amino acid residuesthat interact with an antigen and confer on the Ab its specificity andaffinity for the antigen. The Ab region includes the “framework” aminoacid residues necessary to maintain the proper conformation of theantigen-binding (or “contact”) residues.

As used herein, the term “chimeric antibody” includes monovalent,divalent or polyvalent Igs. A monovalent chimeric Ab is an HL dimerformed by a chimeric H chain associated through disulfide bridges with achimeric L chain. A divalent chimeric Ab is tetramer H₂L₂ formed by twoHL dimers associated through at least one disulfide bridge. A polyvalentchimeric Ab can also be produced, for example, by employing a C_(H)region that aggregates (e.g., from an IgM H chain, termed the μ chain).

The invention also provides for “derivatives” of the mouse mAbs or thechimeric Abs, which term includes those proteins encoded by truncated ormodified genes to yield molecular species functionally resembling the Igfragments. The modifications include, but are not limited to, additionof genetic sequences coding for cytotoxic proteins such as plant andbacterial toxins. The fragments and derivatives can be produced from anyof the hosts of this invention.

Antibodies, fragments or derivatives having chimeric H chains and Lchains of the same or different V region binding specificity, can beprepared by appropriate association of the individual polypeptidechains, as taught, for example by Sears et al., Proc. Natl. Acad. Sci.USA 72:353-357 (1975). With this approach, hosts expressing chimeric Hchains (or their derivatives) are separately cultured from hostsexpressing chimeric L chains (or their derivatives), and the Ig chainsare separately recovered and then associated. Alternatively, the hostscan be co-cultured and the chains allowed to associate spontaneously inthe culture medium, followed by recovery of the assembled Ig, fragmentor derivative.

The antigen binding region of the chimeric Ab (or a human mAb) of thepresent invention is derived preferably from a non-human Ab specific fore.g., uPA/uPAR or uPAR-integrin complex. Preferred sources for the DNAencoding such a non-human Ab include cell lines which produce Ab,preferably hybridomas. Preferred hybridomas are the ATN-615 hybridomacell line (ATCC Accession No. _) and ATN-658 (ATCC Accession No. _)which were produced as described above and whose V regions have thesequences shown above.

Thus, a preferred chimeric Ab (or human Ab) has a V_(L) sequence SEQ IDNO:1 and a V_(H) sequence SEQ ID NO:2 which are the consensus sequencesof mAb ATN-658. The residues of these V regions that are not in the CDRregions may be varied, preferably as conservative substitutions, as longas the V region results in an Ab with the same antigen-specificity andsubstantially the same antigen-binding affinity or avidity, preferablyat least 20% of the affinity or avidity of an Ab wherein the V_(L)sequence is SEQ ID NO:1 and the V_(H) sequence is SEQ ID NO:2. It ispreferred that in this chimeric (or human) Ab, the three CDR regions ofthe V_(L) chain are SEQ ID NO:3, 4 and 5 and the three CDR regions ofthe V_(H) chain are SEQ ID NO:6, 7 and 8.

Another preferred chimeric Ab (or human Ab) has a V_(L) sequence SEQ IDNO:9 and a V_(H) sequence SEQ ID NO:10 which are the consensus sequencesof mAb ATN-615. The residues of these V regions that are not in the CDRregions may be varied, preferably as conservative substitutions, as longas the V region results in an Ab with the same antigen-specificity andsubstantially the same antigen-binding affinity or avidity, preferablyat least 20% of the affinity or avidity of an Ab wherein the V_(L)sequence is SEQ ID NO:9 and the V_(H) sequence is SEQ ID NO:10. It ispreferred that in this chimeric Ab, the three CDR regions of the V_(L)chain are SEQ ID NO:11, 12 and 13 and the three CDR regions of the V_(H)chain are SEQ ID NO:14, 15 and 16.

Preferred nucleic acid molecules for use in constructing a chimeric Ab(or human Ab) of this invention are (a) a nucleic acid molecule with acoding sequence that encodes a V_(L) region with the sequence SEQ IDNO:1 and (b) a nucleic acid molecule with a coding sequence that encodesa V_(H) chain with the sequence SEQ ID NO:2. Also preferred is a nucleicacid molecule that encodes a V_(L) region comprising the three CDRs SEQID NO:3, 4 and 5 and a nucleic acid molecule that encodes a V_(H) regioncomprising the three CDRs SEQ ID NO:6, 7 and 8.

Another set of preferred nucleic acid molecules for use in constructinga chimeric Ab (or human Ab) of this invention are (a) a nucleic acidmolecule with a coding sequence that encodes a V_(L) region with thesequence SEQ ID NO:9 and (b) a nucleic acid molecule with a codingsequence that encodes a V_(H) chain with the sequence SEQ ID NO:10. Alsopreferred is a nucleic acid molecule that encodes a V_(L) regioncomprising the three CDRs SEQ ID NO:11, 12 and 13 and a nucleic acidmolecule that encodes a V_(H) region comprising the three CDRs SEQ IDNO:14, 15 and 16.

Alternatively, the non-human Ab producing cell from which the V regionof the Ab of the invention is derived may be a B lymphocyte obtainedfrom the blood, spleen, lymph nodes or other tissue of an animalimmunized with D2D3 of suPAR. The Ab-producing cell contributing thenucleotide sequences encoding the antigen-binding region of the chimericAb of the present invention may also be produced by transformation of anon-human, such as a primate, or a human cell. For example, a Blymphocyte which produces an Ab specific, e.g., uPA/uPAR oruPAR-integrin complex may be infected and transformed with a virus suchas Epstein-Barr virus to yield an immortal Ab producing cell (Kozbor etal. Immunol. Today 4:72-79 (1983)). Alternatively, the B lymphocyte maybe transformed by providing a transforming gene or transforming geneproduct, as is well-known in the art. Preferably, the antigen bindingregion will be of murine origin. In other embodiments, the antigenbinding region may be derived from other animal species, in particularrodents such as rat or hamster.

The murine or chimeric mAb of the present invention may be produced inlarge quantities by injecting hybridoma or transfectoma cells secretingthe Ab into the peritoneal cavity of mice and, after appropriate time,harvesting the ascites fluid which contains a high titer of the mAb, andisolating the mAb therefrom. For such in vivo production of the mab witha non-murine hybridoma (e.g., rat or human), hybridoma cells arepreferably grown in irradiated or athymic nude mice.

Alternatively, the antibodies may be produced by culturing hybridoma (ortransfectoma) cells in vitro and isolating secreted mAb from the cellculture medium.

Human genes which encode the constant C regions of the chimericantibodies of the present invention may be derived from a human fetalliver library or from any human cell including those which express andproduce human Igs. The human C_(H) region can be derived from any of theknown classes or isotypes of human H chains, including γ, μ, α, δ or ε,and subtypes thereof, such as G1, G2, G3 and G4. Since the H chainisotype is responsible for the various effector functions of an Ab, thechoice of C_(H) region will be guided by the desired effector functions,such as complement fixation, or activity in Ab-dependent cellularcytotoxicity (ADCC). Preferably, the C_(H) region is derived from γ1(IgG1), γ3 (IgG3), γ4 (IgG4), or μ (IgM).

The human C_(L) region can be derived from either human L chain isotype,κ or λ.

Genes encoding human Ig C regions are obtained from human cells bystandard cloning techniques (Sambrook, J. et al., Molecular Cloning: ALaboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989)). Human C region genes are readily available fromknown clones containing genes representing the two classes of L chains,the five classes of H chains and subclasses thereof. Chimeric Abfragments, such as F(ab′)₂ and Fab, can be prepared by designing achimeric H chain gene which is appropriately truncated. For example, achimeric gene encoding an H chain portion of an F(ab′)₂ fragment wouldinclude DNA sequences encoding the CH₁ domain and hinge region of the Hchain, followed by a translational stop codon to yield the truncatedmolecule.

Generally, the chimeric antibodies of the present invention are producedby cloning DNA segments encoding the H and L chain antigen-bindingregions of a specific Ab of the invention, preferably non-human, andjoining these DNA segments to DNA segments encoding human C_(H) andC_(L) regions, respectively, to produce chimeric Ig-encoding genes.

Thus, in a preferred embodiment, a fused gene is created which comprisesa first DNA segment that encodes at least the antigen-binding region ofnon-human origin, such as a functionally rearranged V region withjoining (J) segment, linked to a second DNA segment encoding at least apart of a human C region.

The DNA encoding the Ab-binding region may be genomic DNA or cDNA. Aconvenient alternative to the use of chromosomal gene fragments as thesource of DNA encoding the murine V region antigen-binding segment isthe use of cDNA for the construction of chimeric Ig genes, as reportedby Liu et al. (Proc. Natl. Acad. Sci., USA 84:3439 (1987); J. Immuno.139:3521 (1987), which references are hereby incorporated by reference.The use of cDNA requires that gene expression elements appropriate forthe host cell be combined with the gene in order to achieve synthesis ofthe desired protein. The use of cDNA sequences is advantageous overgenomic sequences (which contain introns), in that cDNA sequences can beexpressed in bacteria or other hosts which lack appropriate RNA splicingsystems.

Therefore, in an embodiment utilizing cDNA encoding the Ab V region, themethod of producing the chimeric Ab involves several steps, outlinedbelow:

-   1. Isolation of messenger RNA (mRNA) from the cell line producing    the mAb, cloning and cDNA production therefrom;-   2. Preparation of a full length cDNA library from purified mRNA from    which the appropriate V region gene segments of the L and H chain    genes can be: (i) identified with appropriate probes, (ii)    sequenced, and (iii) made compatible with a C gene segment;-   3. Preparation of C region gene segments by cDNA preparation and    cloning;-   4. Construction of complete H or L chain coding sequences by linkage    of the cloned specific V region gene segments to cloned human C    region gene, as described above;-   5. Expression and production of chimeric L and H chains in selected    hosts, including prokaryotic and eukaryotic cells.

One common feature of all Ig H and L chain genes and their encoded mRNAsis the J region. H and L chain J regions have different sequences, but ahigh degree of sequence homology exists (greater than 80%) among eachgroup, especially near the C region. This homology is exploited in thismethod and consensus sequences of H and L chain J regions may be used todesign oligonucleotides for use as primers for introducing usefulrestriction sites into the J region for subsequent linkage of V regionsegments to human C region segments.

C region cDNA vectors prepared from human cells can be modified bysite-directed mutagenesis to place a restriction site at the analogousposition in the human sequence. For example, one can clone the completehuman κ chain C (C_(k)) region and the complete human γ-1 C region(C_(γ-1)). In this case, the alternative method based upon genomic Cregion clones as the source for C region vectors would not allow thesegenes to be expressed in bacterial systems where enzymes needed toremove intervening sequences are absent. Cloned V region segments areexcised and ligated to L or H chain C region vectors. Alternatively, thehuman C_(γ-1) region can be modified by introducing a termination codonthereby generating a gene sequence which encodes the H chain portion ofa Fab molecule. The coding sequences with linked V and C regions arethen transferred into appropriate expression vehicles for expression inappropriate hosts, prokaryotic or eukaryotic.

Two coding DNA sequences are said to be “operably linked” if the linkageresults in a continuously translatable sequence without alteration orinterruption of the triplet reading frame. A DNA coding sequence isoperably linked to a gene expression element if the linkage results inthe proper function of that gene expression element to result inexpression of the coding sequence.

Expression vehicles include plasmids or other vectors. Preferred amongthese are vehicles carrying a functionally complete human C_(H) or C_(L)chain sequence having appropriate restriction sites engineered so thatany V_(H) or V_(L) chain sequence with appropriate cohesive ends can beeasily inserted therein. Human C_(H) or C_(L) chain sequence-containingvehicles thus serve as intermediates for the expression of any desiredcomplete H or L chain in any appropriate host.

A chimeric mouse-human Ab will typically be synthesized from genesdriven by the chromosomal gene promoters native to the mouse H and Lchain V regions used in the constructs. Splicing usually occurs betweenthe splice donor site in the mouse J region and the splice acceptor sitepreceding the human C region and also at the splice regions that occurwithin the human C_(H) region; polyadenylation and transcriptiontermination occur at native chromosomal sites downstream of the humancoding regions.

Gene expression elements useful for the expression of cDNA genesinclude: (a) viral transcription promoters and their enhancer elements,such as the SV40 early promoter (Okayama, H. et al., Mol. Cell. Biol.3:280 (1983)), Rous sarcoma virus LTR (Gorman, C. et al., Proc. Natl.Acad. Sci., USA 79:6777 (1982)), and Moloney murine leukemia virus LTR(Grosschedl, R et al., Cell 41:885 (1985)); (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region(Okayama et al., supra); and (c) polyadenylation sites such as in SV40(Okayama et al., supra).

Ig cDNA genes may be expressed as described by Liu et al., supra, andWeidle, U H et al., Gene 51:21-29 (1987), using as expression elementsthe SV40 early promoter and its enhancer, the mouse Ig H chain promoterenhancers, SV40 late region mRNA splicing, rabbit β-globin interveningsequence, Ig and rabbit β-globin polyadenylation sites, and SV40polyadenylation elements. For Ig genes comprised of part cDNA, partgenomic DNA (Whittle, N et al., Protein Eng. 1:499-505 (1987)), thetranscriptional promoter is human cytomegalovirus, the promoterenhancers are cytomegalovirus and mouse/human Ig, and mRNA splicing andpolyadenylation regions are from the native chromosomal Ig sequences. Inone embodiment, for expression of cDNA genes in rodent cells, thetranscriptional promoter is a viral LTR sequence, the transcriptionalpromoter enhancers are either or both the mouse Ig H chain enhancer andthe viral LTR enhancer, the splice region contains an intron of greaterthan 31 bp, and the polyadenylation and transcription terminationregions are derived from the native chromosomal sequence correspondingto the Ig chain being synthesized. In other embodiments, cDNA sequencesencoding other proteins are combined with the above-recited expressionelements to achieve expression of the proteins in mammalian cells.

Each fused gene is assembled in, or inserted into, an expression vector.Recipient cells capable of expressing the chimeric Ig chain gene productare then transfected singly with a chimeric H or chimeric Lchain-encoding gene, or are co-transfected with a chimeric H and achimeric L chain gene. The transfected recipient cells are culturedunder conditions that permit expression of the incorporated genes andthe expressed Ig chains or intact antibodies or fragments are recoveredfrom the culture. In one embodiment, the fused genes encoding thechimeric H and L chains, or portions thereof, are assembled in separateexpression vectors that are then used to co-transfect a recipient cell.

Each vector may contain two selectable genes, a first selectable genedesigned for selection in a bacterial system and a second selectablegene designed for selection in a eukaryotic system, wherein each vectorhas a different pair of genes. This strategy results in vectors whichfirst direct the production, and permit amplification, of the fusedgenes in a bacterial system. The genes so produced and amplified in abacterial host are subsequently used to co-transfect a eukaryotic cell,and allow selection of a co-transfected cell carrying the desiredtransfected genes. Examples of selectable genes for use in a bacterialsystem are the gene that confers resistance to ampicillin and the genethat confers resistance to chloramphenicol. Preferred selectable genesfor use in eukaryotic transfectants include the xanthine guaninephosphoribosyl transferase gene (designated gpt) and thephosphotransferase gene from Tn5 (designated neo).

Selection of cells expressing gpt is based on the fact that the enzymeencoded by this gene utilizes xanthine as a substrate for purinenucleotide synthesis, whereas the analogous endogenous enzyme cannot. Ina medium containing (1) mycophenolic acid, which blocks the conversionof inosine monophosphate to xanthine monophosphate (XMP), and (2)xanthine, only cells expressing the gpt gene can survive. The product ofthe neo gene blocks the inhibition of protein synthesis by theantibiotic G418 and other antibiotics of the neomycin class.

The two selection procedures can be used simultaneously or sequentiallyto select for the expression of Ig chain genes introduced on twodifferent DNA vectors into a eukaryotic cell. It is not necessary toinclude different selectable markers for eukaryotic cells; an H and an Lchain vector, each containing the same selectable marker can beco-transfected. After selection of the appropriately resistant cells,the majority of the clones will contain integrated copies of both H andL chain vectors.

Alternatively, the fused genes encoding the chimeric H and L chains canbe assembled on the same expression vector.

For transfection of the expression vectors and production of thechimeric Ab, the preferred recipient cell line is a myeloma cell.Myeloma cells can synthesize, assemble and secrete Igs encoded bytransfected Ig genes and possess the mechanism for glycosylation of theIg. A particularly preferred recipient cell is the Ig-non-producingmyeloma cell SP2/0 (ATCC #CRL 8287). SP2/0 cells produce only Ig encodedby the transfected genes. Myeloma cells can be grown in culture or inthe peritoneal cavity of a mouse, where secreted Ig can be obtained fromascites fluid. Other suitable recipient cells include lymphoid cellssuch as B lymphocytes of human or non-human origin, hybridoma cells ofhuman or non-human origin, or interspecies heterohybridoma cells.

The expression vector carrying a chimeric Ab construct of the presentinvention may be introduced into an appropriate host cell by any of avariety of suitable means, including such biochemical means astransformation, transfection, conjugation, protoplast fusion, calciumphosphate-precipitation, and application with polycations such asdiethylaminoethyl (DEAE) dextran, and such mechanical means aselectroporation, direct microinjection, and microprojectile bombardment.

The chimeric Ig coding sequences or genes of the present invention canalso be expressed in nonlymphoid mammalian cells or in other eukaryoticcells, such as yeast, or in prokaryotic cells, in particular bacteria.Yeast provides substantial advantages over bacteria for the productionof Ig H and L chains. Yeasts carry out post-translational peptidemodifications including glycosylation. A number of recombinant DNAstrategies now exist which utilize strong promoter sequences and highcopy number plasmids which can be used for production of the desiredproteins in yeast. Yeast recognizes leader sequences of cloned mammaliangene products and secretes peptides bearing leader sequences (i.e.,pre-peptides). Yeast gene expression systems can be routinely evaluatedfor the levels of production, secretion and the stability of chimeric Hand L chain proteins and assembled chimeric Abs. Any of a series ofyeast gene expression systems incorporating promoter and terminationelements from the actively expressed genes coding for glycolytic enzymesproduced in large quantities when yeasts are grown in media rich inglucose can be utilized. Known glycolytic genes can also provide veryefficient transcription control signals. For example, the promoter andterminator signals of the phosphoglycerate kinase (PGK) gene can beutilized. A number of approaches may be taken for evaluating optimalexpression plasmids for the expression of cloned Ig cDNAs in yeast (seeGlover, D. M., ed., DNA Cloning, IRL Press, 1985).

Bacterial strains may also be utilized as hosts for the production of Abmolecules or Ab fragments described by this invention, E. coli K12strains such as E. coli W3110 (ATCC# 27325), and other enterobacteriasuch as Salmonella typhimurium or Serratia marcescens, and variousPseudomonas species may be used.

Plasmid vectors containing replicon and control sequences which arederived from species compatible with a host cell are used in connectionwith these bacterial hosts. The vector carries a replication site, aswell as specific genes which are capable of providing phenotypicselection in transformed cells. A number of approaches may be taken forevaluating the expression plasmids for the production of chimeric Abs orAb chains encoded by the cloned Ig cDNAs in bacteria (see Glover,supra).

Preferred hosts are mammalian cells, grown in vitro or in vivo.Mammalian cells provide post-translational modifications to Ig proteinmolecules including leader peptide removal, folding and assembly of Hand L chains, glycosylation of the Ab molecules, and secretion offunctional Ab protein. Mammalian cells which may be useful as hosts forthe production of Ab proteins, in addition to the cells of lymphoidorigin described above, include cells of fibroblast origin, such as Vero(ATCC CRL 81) or CHO-K1 (ATCC CRL 61). Many vector systems are availablefor the expression of cloned H and L chain genes in mammalian cells (seeGlover, supra). Different approaches can be followed to obtain completeH₂L₂ Abs.

For in vivo use, particularly for injection into humans, it is desirableto decrease the immunogenicity of the mAb by making mouse-human (orrodent-human) chimeric Abs as above, or by humanizing the Abs usingmethods known in the art. The humanized Ab may be the product of ananimal having transgenic human Ig Constant region genes (see for exampleWO90/10077 and WO90/04036). Alternatively, the Ab of interest may begenetically engineered to substitute the CH₁, CH₂, CH₃, hinge domains,and/or the framework domain with the corresponding human sequence (seeWO92/02190).

Single Chain Antibodies

The Ab of the present invention may be produced as a single chain Ab orscFv instead of the normal multimeric structure. Single chain Absinclude the hypervariable regions from an Ig of interest and recreatethe antigen binding site of the native Ig while being a fraction of thesize of the intact Ig (Skerra, A. et al. (1988) Science, 240: 1038-1041;Pluckthun, A. et al. (1989) Methods Enzymol. 178: 497-515; Winter, G. etal. (1991) Nature, 349: 293-299); Bird et al., (1988) Science 242:423;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879; Jost C R etal., J Biol Chem. 1994 269:26267-26273; U.S. Pat. Nos. 4,704,692,4,853,871, 4,94,6778, 5,260,203, 5,455,030). DNA sequences encoding theV regions of the H chain and the L chain are ligated to a linkerencoding at least about 4 amino acids (typically small neutral aminoacids). The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original Ab.

One method of producing the Abs of the present invention is to link twoor more peptides or polypeptides together by protein chemistrytechniques. For example, peptides or polypeptides can be chemicallysynthesized using currently available laboratory equipment using eitherFmoc (9-fluorenylmethyloxycarbonyl) or tBoc (tert -butyloxycarbonyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to an Ab chain or antigen-binding fragment thereof can besynthesized by standard chemical reactions. For example, a peptide orpolypeptide can be synthesized but not cleaved from its synthesis resinwhereas the other fragment of an Ab can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. By peptide condensationreactions, these two fragments can be covalently joined via a peptidebond at their C- and N-termini, respectively, to form an Ab, or afragment thereof. (Grunt, G A, Synthetic Peptides: A User Guide, W.H.Freeman and Co., N.Y. (1992); Bodansky, M et al., eds, Principles ofpeptide Synthesis, Springer-Verlag Inc., N.Y. (1993))

Antibodies can be selected for particular desired properties. In thecase of an Ab to be used in vivo, Ab screening procedures can includeany of the in vitro or in vivo bioassays that measure binding to e.g.,uPA/uPAR or uPAR-integrin complex, to cells expressing the relevantpolypeptide or peptide epitope. Moreover, the Abs may be screened invarious of tumor models such as a xenogeneic mouse model in which ahuman tumor cell line expressing the antigen is grown inimmunocompromised, e.g., nude, mice.

Diagnostically Labeled Antibody

The term “diagnostically labeled” means that the present Ab has attachedto it a diagnostically detectable label. There are many different labelsand methods of labeling known to those of ordinary skill in the art,described below. General classes of labels which can be used in thepresent invention include radioactive isotopes, paramagnetic isotopes,and compounds which can be imaged by positron emission tomography (PET),fluorescent or colored compounds, etc. Suitable detectable labelsinclude radioactive, fluorescent, fluorogenic, chromogenic, or otherchemical labels. Useful radiolabels (radionuclides), which are detectedsimply by gamma counter, scintillation counter or autoradiographyinclude ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C. ¹³¹I is also a useful therapeuticisotope (see below).

A number of U.S. patents, incorporated by reference herein, disclosemethods and compositions for complexing metals to larger molecules,including description of useful chelating agents. The metals arepreferably detectable metal atoms, including radionuclides, and arecomplexed to proteins and other molecules. These documents include: U.S.Pat. Nos. 5,627,286; 5,618,513; 5,567,408; 5,443,816; and 5,561,220.

Common fluorescent labels include fluorescein, rhodamine, dansyl,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. The fluorophore, such as the dansyl group, must beexcited by light of a particular wavelength to fluoresce. See, forexample, Haugland, Handbook of Fluorescent Probes and ResearchChemicals, Sixth Ed., Molecular Probes, Eugene, Oreg., 1996).Fluorescein, fluorescein derivatives and fluorescein-like molecules suchas Oregon Green™ and its derivatives, Rhodamine Green™ and RhodolGreen™, are coupled to amine groups using the isothiocyanate,succinimidyl ester or dichlorotriazinyl-reactive groups. Similarly,fluorophores may also be coupled to thiols using maleimide,iodoacetamide, and aziridine-reactive groups. The long wavelengthrhodamines, which are basically Rhodamine Green™ derivatives withsubstituents on the nitrogens, are among the most photostablefluorescent labeling reagents known. Their spectra are not affected bychanges in pH between 4 and 10, an important advantage over thefluoresceins for many biological applications. This group includes thetetramethylrhodamines, X-rhodamines and Texas Red™ derivatives. Otherpreferred fluorophores for derivatizing the peptide according to thisinvention are those which are excited by ultraviolet light. Examplesinclude cascade blue, coumarin derivatives, naphthalenes (of whichdansyl chloride is a member), pyrenes and pyridyloxazole derivatives.Also included as labels are two related inorganic materials that haverecently been described: semiconductor nanocrystals, comprising, forexample, cadmium sulfate (Bruchez, M et al., Science 281:2013-2016(1998), and quantum dots, e.g., zinc-sulfide-capped Cd selenide (Chan, WC et al., Science 281:2016-2018 (1998)).

In yet another approach, the amino group of the Ab is allowed to reactwith reagents that yield fluorescent products, for example,fluorescamine, dialdehydes such as o-phthaldialdehyde,naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.7-nitrobenz-2-oxa-1,3-diazole (NBD) derivatives, both chloride andfluoride, are useful to modify amines to yield fluorescent products.

The Ab of the invention can also be labeled for detection usingfluorescence-emitting metals such as ¹⁵²Eu, or others of the lanthanideseries. These metals can be attached to the peptide using such metalchelating groups as diethylenetriaminepentaacetic acid (DTPA, seeExample X, infra) or ethylene-diaminetetraacetic acid (EDTA). DTPA, forexample, is available as the anhydride, which can readily modify theNH₂-containing peptides of this invention.

For in vivo diagnosis or therapy, radionuclides may be bound to the Abeither directly or indirectly using a chelating agent such as DTPA andDOTA. Examples of such radionuclides are ⁹⁹Tc, ¹²³I, ¹²⁵I, ¹³¹I, ¹¹¹In,⁹⁷Ru, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁰Y and ²⁰¹Tl. Generally, the amountof labeled Ab needed for detectability in diagnostic use will varydepending on considerations such as age, condition, sex, and extent ofdisease in the patient, contraindications, if any, and other variables,and is to be adjusted by the individual physician or diagnostician.Dosage can vary from 0.001 mg/kg to 100 mg/kg.

The Ab can also be made detectable by coupling to a phosphorescent or achemiluminescent compound. The presence of the chemiluminescent-taggedpeptide is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescers are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salt and oxalate ester.Likewise, a bioluminescent compound may be used to label the peptides.Bioluminescence is a type of chemiluminescence found in biologicalsystems in which a catalytic protein increases the efficiency of thechemiluminescent reaction. The presence of a bioluminescent protein isdetermined by detecting the presence of luminescence. Importantbioluminescent compounds for purposes of labeling are luciferin,luciferase and acquorin.

In yet another embodiment, colorimetric detection is used, based onchromogenic compounds which have, or result in, chromophores with highextinction coefficients.

In situ detection of the labeled peptide may be accomplished by removinga histological specimen from a subject and examining it by microscopyunder appropriate conditions to detect the label. Those of ordinaryskill will readily perceive that any of a wide variety of histologicalmethods (such as staining procedures) can be modified in order toachieve such in situ detection.

For diagnostic in vivo radioimaging, the type of detection instrumentavailable is a major factor in selecting a radionuclide. Theradionuclide chosen must have a type of decay which is detectable by aparticular instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention. Another factor in selecting a radionuclide for in vivodiagnosis is that its half-life be long enough so that the label isstill detectable at the time of maximum uptake by the target tissue, butshort enough so that deleterious irradiation of the host is minimized.In one preferred embodiment, a radionuclide used for in vivo imagingdoes not emit particles, but produces a large number of photons in a140-200 keV range, which may be readily detected by conventional gammacameras.

In vivo imaging may be used to detect occult metastases which are notobservable by other methods. Imaging could be used, for example, tostage tumors non-invasively.

Use of Antibodies to Detect uPA- or uPAR-Complexes by Immunoassay

Antibodies of this invention are useful in immunoassays to detectmolecules containing these epitopes in tissue sample or a body fluid,such as serum or plasma. Such Abs would detect the antigen or anepitope-bearing fragment thereof. Thus, if proteolysis in the tumormilieu results in release of the fragments or in tissue.

Any conventional immunoassay known in the art may be employed for thispurpose, though Enzyme Immunoassays such as ELISA are preferred.Immunoassay methods are also described in references cited above.

Competitive immunoassays are typically used to detect molecules in atest sample that are ligands for the complex that may mimic the mAbs intheir binding specificity, affinity, capacity, etc. In one embodiment acompetitive binding assay, the amount of Ab bound to the complex ismeasured (directly or indirectly using a labeled anti-Ig). Competition(i.e., less binding of Ab to complex) in the presence of the test sampleis evidence that one or more components of the sample bind to thecomplex. It is expected that most compounds being tested will bind withmoderate affinities (approximately 1-10 μM)

In another embodiment, a solid support, e.g., a microplate, is coatedwith the mAb of interest. The test sample is added and incubated, e.g.,for about 30 minutes to allow binding of relevant molecules to the Ab.The plates are washed and the complex, in detectably labeled form (e.g.,biotinylated), is added as the competitive ligand, and allowed tocompete with the test sample for binding to the Ab. A “positive” resultfor the test sample will be expressed as less binding of labeled complexbound to the solid phase. This approach, in which the complex solutionand sample solution are not added simultaneously, avoids the confoundingeffects of test sample binding directly to the complex, because any testsample present must first be captured by the immobilized mAb.Preferably, to assure that binding is specific, a series of dilutionsare run to obtain a dilution curve. This will show if, for example,there is 50% less binding/signal ratio with half the sample. In theabsence of such dilution effects, it may be concluded that multiplebinding entities are entering into the assay. Results are more rigorousif molecules binding at the mAb binding site have similar affinities.

Immunohistochemical Assays

One preferred assay for detecting the antigens in a tissue is byimmunohistochemistry, using any conventional assay methods, with whichthe art is replete. A preferred assay is the one described in theExamples below. For a description of such methods, see, for example,Dabbs, D J, Diagnostic Immunohistochemistry, Churchill Livingstone,2001, which is incorporated by reference in its entirety.

Non-Histological Immunoassays

Preferred immunoassays are enzyme immunoassays (EIA's) such as ELISA,which employ antigens or Abs immobilized to solid supports. For thepresent compositions and methods, the solid support is preferably anyone of polystyrene, polypropylene, polyethylene, dextran, nylon,polyacrylamide, polyvinylidene difluoride, natural cellulose, modifiedcellulose, nitrocellulose, agarose and magnetic beads. In a preferredembodiment, the surface of polystyrene or other plastic multiwell platesserves as the solid support. In another embodiment, a solid support towhich the Ab or antigen is affixed to the bottom or placed loosely inthe wells of multiwell plates. Multiwell plates in which the bottoms ofthe wells comprise nitrocellulose or a similar membrane material andthrough which liquid can be moved under pressure or vacuum may also beused.

Typical, and preferred, immunoassays include “forward” assays in whichthe Ab immobilized to a solid support is first contacted with the samplebeing tested to bind or “extract” the antigen from the sample byformation of a binary immobilized Ab-antigen complex. After suitableincubation, the solid support is washed to remove the residue of thefluid sample including unbound antigen, if any, and then contacted withthe solution containing an unknown quantity of labeled Ab (whichfunctions as a “reporter molecule”). After a second incubation, thatpermits the labeled Ab to complex with the immobilized antigen throughthe unlabeled Ab, the solid support is washed a second time to removethe unreacted labeled Ab and the immobilized label is measured. Thistype of forward sandwich assay may be a simple “yes/no” assay todetermine whether antigen is present or may be made quantitative bycomparing the amount of immobilized labeled Ab with the amountimmobilized when a standard sample containing a known quantity ofantigen is used.

So called “simultaneous” and “reverse” sandwich assays may also be used.A simultaneous assay involves a single incubation step as theimmobilized Ab and labeled Ab are added simultaneously to the sample.After appropriate incubation, the solid support is washed to removeresidue of the sample and uncomplexed labeled Ab. The presence or amountof labeled Ab associated with the solid support is then determined as inthe above conventional “forward” sandwich assay.

In a “reverse” assay, a solution of labeled Ab is added to the sampleafter a suitable incubation period followed by addition of immobilizedunlabeled Ab. After a second incubation, the solid phase material iswashed in conventional fashion to free it of the residue of the sampleand unreacted labeled Ab. The determination of immobilized Ab associatedwith the solid support is then determined as in the “simultaneous” and“forward” assays.

Assay for Antibody Binding to uPAR on Whole Cells

The uPAR-targeting Ab and/or conjugate thereof is readily tested forbinding to uPAR, preferably by measuring inhibition of the binding of[¹²⁵I]DFP-uPA to uPAR in a competitive ligand-binding assay or bydirectly labeling the Ab with [¹²⁵I]. The assay may employ whole cellsthat express uPAR, for example cells lines such as A2780 or HeLa. Apreferred assay is conducted as follows. Cells (about 5×10⁴/well) areplated in medium (e.g., MEM with Earle's salts/10% FBS+antibiotics) in24-well plates, then incubated in a humid 5% CO₂ atmosphere until thecells reach 70% confluence. Catalytically inactivated high molecularweight uPA (DFP-uPA) is radioiodinated using Iodo-gen® (Pierce) to aspecific activity of about 250,000 cpm/μg. The cell-containing platesare then chilled on ice and the cells are washed twice (5 minutes each)with cold PBS/0.05% Tween-80. Test Abs and/or conjugates thereof areserially diluted in cold PBS/0.1% BSA/0.01% Tween-80 and added to eachwell to a final volume of 0.3 mL 10 minutes prior to the addition of the[¹²⁵I]DFP-uPA. Each well then receives 9500 cpm of [¹²⁵I]DFP-uPA at afinal concentration of 0.2 nM). The plates are then incubated at 4° C.for 2 hrs, after which time the cells are washed 3× (5 minutes each)with cold PBS/0.05% Tween-80. NaOH (1N) is added to each well in 0.5 mLto lyse the cells, and the plate is incubated for 5 minutes at roomtemperature or until all the cells in each well are lysed as determinedby microscopic examination. The contents of each well are then aspiratedand the total counts in each well determined using a gamma counter. Eachcompound is tested in triplicate and the results are expressed as apercentage of the total radioactivity measured in wells containing[¹²⁵I]DFP-uPA alone, which is taken to represent maximum (100%) binding.

The inhibition of binding of [¹²⁵I]DFP-uPA to uPAR is usuallydose-related, such that the concentration of the test compound necessaryto produce a 50% inhibition of binding (the IC₅₀ value), which isexpected to fall in the linear part of the curve, is easily determined.In general, Abs and/or conjugates thereof have IC₅₀ values of less thanabout 10⁻⁵ M. Preferably, Abs and/or conjugates thereof have IC₅₀ valuesof less than about 10⁻⁶ M, more preferably, less than about 10⁻⁷M.

Assays of Biological Activity of Anti-uPAR Antibodies or Other Ligands

Those of skill in the art will appreciate that the in vitro and in vivoassays useful for measuring the activity of the Abs or otheruPAR-binding ligands of the invention or of conjugates thereof, asdescribed herein, are intended to be illustrative and neithercomprehensive nor limiting.

Assay for EC migration

For EC migration studies, transwells are coated with type I collagen (50μg/nl) by adding 200 μL of the collagen solution per transwell, thenincubating overnight at 37° C. The transwells are assembled in a 24-wellplate and a chemoattractant (e.g., FGF-2) is added to the bottom chamberin a total volume of 0.8 mL media. ECs, such as human umbilical veinendothelial cells (HUVEC), which have been detached from monolayerculture using trypsin, are diluted to a final concentration of about 10⁶cells/mL with serum-free media and 0.2 mL of this cell suspension isadded to the upper chamber of each transwell. Inhibitors to be testedmay be added to both the upper and lower chambers and the migration isallowed to proceed for 5 hrs in a humidified atmosphere at 37° C. Thetranswells are removed from the plate stained using DiffQuik®. Cellswhich did not migrate are removed from the upper chamber by scrapingwith a cotton swab and the membranes are detached, mounted on slides,and counted under a high-power field (400×) to determine the number ofcells migrated.

Biological Assay of Anti-Invasive Activity

The ability of cells such as ECs or tumor cells (e.g., PC-3 humanprostatic carcinoma cells) to invade through a reconstituted basementmembrane (Matrigel®) in an assay known as a Matrigel® invasion assaysystem is well known (Kleinman et al., Biochemistry 1986, 25: 312-318;Parish et al., 1992, Int. J. Cancer 52:378-383). Matrigel® is areconstituted basement membrane containing type IV collagen, laminin,heparan sulfate proteoglycans such as perlecan (which bind to andlocalize bFGF), vitronectin as well as transforming growth factor-β(TGFβ), urokinase-type plasminogen activator (uPA), tissue plasminogenactivator (tPA) and the serpin known as plasminogen activator inhibitortype 1 (PAI-1) (Chambers et al., Canc. Res. 1995, 55:1578-1585). It isaccepted in the art that results obtained in this type of assay for Absand/or conjugates thereof or other ligands which target extracellularreceptors or enzymes are predictive of the efficacy of these Abs and/orconjugates thereof in vivo (Rabbani et al., Int. J. Cancer 1995, 63:840-845).

Such assays employ transwell tissue culture inserts. Invasive cells aredefined as cells which traverse through the Matrigel® and upper aspectof a polycarbonate membrane and adhere to the bottom of the membrane.Transwells (e.g., from Costar) containing polycarbonate membranes (8.0μm pore size) are coated with Matrigel® (e.g., from CollaborativeResearch), which has been diluted in sterile PBS to a finalconcentration of about 75 μg/mL (e.g., 60 μL of diluted Matrigel® perinsert), and placed in the wells of a 24-well plate. The membranes aredried overnight in a biological safety cabinet, then rehydrated byadding 100 μL of medium, e.g., DMEM, supplemented with antibiotics for 1hour on a shaker table. The DMEM is removed from each insert byaspiration and 0.8 mL of complete DMEM (+/10% FBS and antibiotics) isadded to each well of the 24-well plate such that it surrounds theoutside of the transwell (“lower chamber”). Fresh DMEM with antibiotics(100 μL), human Glu-plasminogen (5 μg/mL), and any inhibitors to betested are added to the top, inside of the transwell (“upper chamber”).The cells which are to be tested are trypsinized and resuspended inDMEM+antibiotics and added to the top chamber of the transwell at afinal concentration of about 8×10⁵ cells/mL. The final volume of theupper chamber is adjusted to 200 μL. The assembled plate is thenincubated in a humid 5% CO₂ atmosphere for about 72 hours. Afterincubation, the cells are fixed and stained using DiffQuik® (Giemsastain) and the upper chamber is then scraped using a cotton swab toremove the Matrigel® and any cells which did not invade through themembrane. The membranes are detached from the transwell using an X-acto®blade, mounted on slides using Permount® and coverslips, then countedunder a microscope using high power (e.g., 400×). A mean number ofinvading cells from 5-10 counted fields is calculated and plotted as afunction of inhibitor concentration.

Tube-Formation Assays of Anti-Angiogenic Activity

ECs, for example, human umbilical vein endothelial cells (HUVEC) orhuman microvascular endothelial cells (HMVEC) which can be prepared orobtained commercially, are mixed at a concentration of 2×10⁵ cells/mLwith fibrinogen (5 mg/mL in phosphate buffered saline (PBS) in a 1:1(v/v) ratio. Thrombin is added (5 units/mL final concentration) and themixture is immediately transferred to a 24-well plate (0.5 mL per well).The fibrin gel is allowed to form and then VEGF and bFGF are added tothe wells (each at 5 mg/mL final concentration) along with the testcompound. The cells are incubated at 37° C. in 5% CO₂ for 4 days atwhich time the cells in each well are counted and classified as eitherrounded, elongated with no branches, elongated with one branch, orelongated with 2 or more branches. Results are expressed as the averageof 5 different wells for each concentration of compound. Typically, inthe presence of angiogenic inhibitors, cells remain either rounded orform undifferentiated tubes (e.g. 0 or 1 branch). This assay isrecognized in the art to be predictive of angiogenic (oranti-angiogenic) efficacy in vivo (Min et al., Cancer Res. 1996, 56:2428-2433).

In an alternate assay, EC tube formation is observed when ECs arecultured on Matrigel® (Schnaper H W et al., J. Cell. Physiol. 1995,165:107-118). 10⁴ EC /well are transferred onto Matrigel®-coated 24-wellplates, and tube formation is quantitated after 48 hrs. Inhibitors aretested by adding them either at the time of adding the ECs or at varioustime points thereafter. Tube formation can also be stimulated by adding(a) an angiogenic growth factor such as bFGF or VEGF, (b) adifferentiation stimulating agent (e.g., PMA) or (c) a combination ofthese.

While not wishing to be bound by theory, this assay models angiogenesisby presenting to the ECs a particular type of basement membrane, namelythe layer of matrix which migrating and differentiating ECs would beexpected to encounter first. In addition to bound growth factors, thematrix components found in Matrigel® (and in basement membranes insitu), or proteolytic products thereof, may also be stimulatory for ECtube formation which makes this model complementary to the fibrin gelangiogenesis model previously described (Blood, C H et al., Biochim.Biophys. Acta 1990, 1032:89-118; Odedra, R et al., Pharmac. Ther. 1991,49:111-124).

Assays for Inhibition of Cell Proliferation

The ability of the Abs and/or conjugates of this invention to inhibitthe proliferation of ECs may be determined in a 96-well format. Type Icollagen (gelatin) is used to coat the wells of the plate (0.1-1 mg/mLin PBS, 0.1 mL per well for 30 minutes at room temperature). Afterwashing the plate (3× using PBS), 3-6×10³ cells are plated per well andallowed to attach for 4 hrs (37° C./5% CO₂) in Endothelial Growth Medium(EGM; Clonetics) or M199 medium supplemented with 0.1-2% FBS. The mediumand any unattached cells are removed at the end of 4 hrs and freshmedium supplemented with bFGF (1-10 ng/mL) or VEGF (1-10 ng/mL) is addedto each well. Antibodies and/or conjugates to be tested are added last,and the plate is allowed to incubate (37° C./5% CO₂) for 24-48 hrs. Thechromogenic compound MTS (Promega) is added to each well and allowed toincubate from 1-4 hrs. The color developing in each well is directlyproportional to the cell number, thereby serving as a surrogate forcounting cells. Absorbance read at 490 nm is used to determine thedifferences in cell numbers, i.e., proliferation, between control wellsand those containing test Abs and/or conjugates.

A similar assay employing cultured adherent tumor cells may also beused. However, collagen may be omitted in this format. Tumor cells(e.g., 3-10×10³/well) are plated and allowed to adhere overnight.Serum-free medium is then added, and the cells forced to synchronize for24 hrs. Medium+10% FBS is then added to each well to stimulateproliferation. Antibodies and/or conjugates to be tested are included insome of the wells. After 24 hrs, MTS is added to the plate and the assaydeveloped and read as above.

Assays of Cytotoxicity

The anti-proliferative and cytotoxic effects of Abs and/or conjugatesthereof may be determined for various cell types including tumor cells,ECs, fibroblasts and macrophages. This is especially useful when testinga Ab which has been conjugated to a therapeutic moiety such as aradiotherapeutic or a toxin. For example, a conjugate of one of the Absof the invention with Bolton-Hunter reagent which has been iodinatedwith ¹³¹I would be expected to inhibit the proliferation of cellsexpressing uPAR (most likely by inducing apoptosis). Anti-proliferativeeffects would be expected against tumor cells and stimulated endothelialcells but, under some circumstances not quiescent endothelial cells ornormal human dermal fibroblasts. Any anti-proliferative or cytotoxiceffects observed in the normal cells may represent non-specific toxicityof the conjugate.

A typical assay would involve plating cells at a density of 5-10,000cells per well in a 96-well plate. The compound to be tested is added ata concentration 10× the IC₅₀ measured in a binding assay (this will varydepending on the conjugate) and allowed to incubate with the cells for30 minutes. The cells are washed 3× with media, then fresh mediacontaining [³H]thymidine (1 μCi/mL) is added to the cells and they areallowed to incubate at 37° C. in 5% CO₂ for 24 and 48 hours. Cells arelysed at the various time points using 1 M NaOH and counts per welldetermined using a β-counter. Proliferation may be measurednon-radioactively using MTS reagent or CyQuant® to measure total cellnumber. For cytotoxicity assays (measuring cell lysis), a Promega96-well cytotoxicity kit is used. If there is evidence ofanti-proliferative activity, induction of apoptosis may be measuredusing TumorTACS (Genzyme).

Assay of Caspase-3 Activity

The ability of the Abs and/or conjugates to promote apoptosis of EC'smay be determined by measuring activation of caspase-3. Type I collagen(gelatin) is used to coat a P100 plate and 5×10⁵ ECs are seeded inEGM+10% FBS. After 24 hours (at 37° C./5% CO₂) the medium is replaced byEGM+2% FBS, 10 ng/ml bFGF and the desired test compound. The cells areharvested after 6 hrs, cell lysates prepared in 1% Triton X-100detergent, and the lysates assayed using the EnzChek®Caspase-3 Assay Kit#1 (Molecular Probes) according to the manufactures' instructions.

Corneal Angiogenesis Model

The protocol used is essentially identical to that described by Volpert,O V et al., J. Clin. Invest. 1996, 98:671-679. Briefly, female Fischerrats (120-140 gms) are anesthetized and pellets (5 μl) comprised ofHydron®, bFGF (150 nM), and the Abs and/or conjugates thereof to betested are implanted into tiny incisions made in the cornea 1.0-1.5 mmfrom the limbus. Neovascularization is assessed at 5 and 7 days afterimplantation. On day 7, animals are anesthetized and infused with a dyesuch as colloidal carbon to stain the vessels. The animals are theneuthanized, the corneas fixed with formalin, and the corneas flattenedand photographed to assess the degree of neovascularization. Neovesselsmay be quantitated by imaging the total vessel area or length or simplyby counting vessels.

Chick Chorioallantoic Membrane (CAM) Angiogenesis Assay

This assay is performed essentially as described by Nguyen et al.,Microvascular Res. 1994, 47:3140. A mesh containing either angiogenicfactors (bFGF) or tumor cells plus a test compound, here the anti-uPARAbs or conjugates, placed onto the CAM of an 8-day old chick embryo andthe CAM observed for 3-9 days after implantation of the sample.Angiogenesis is quantitated by determining the percentage of squares inthe mesh which contain visible blood vessels.

Matrigel® Plug Assay

This assay is performed essentially as described by Passaniti, A et al.,1992, Lab Invest. 67:519-528. Ice-cold Matrigel® (e.g., 500 μL)(Collaborative Biomedical Products, Inc., Bedford, Mass.) is mixed withheparin (e.g., 50 ng/ml), FGF-2 (e.g., 400 ng/ml) and the compound to betested. In some assays, bFGF may be substituted with tumor cells as theangiogenic stimulus. The Matrigel® mixture is injected subcutaneously(s.c.) into 4-8 week-old athymic nude mice at sites near the abdominalmidline, preferably 3 injections per mouse. The injected Matrigel® formsa palpable solid gel. Injection sites are chosen such that each animalreceives a positive control plug (such as FGF2+heparin), a negativecontrol plug (e.g., buffer+heparin) and a plug that includes thecompound being tested for its effect on angiogenesis, e.g.,(FGF-2+heparin+compound). All treatments groups are preferably run intriplicate. Animals are sacrificed by cervical dislocation at about 7days post injection or another time that may be optimal for observingangiogenesis. The mouse skin is detached along the abdominal midline,and the Matrigel® plugs are recovered and scanned microscopicallyimmediately at high resolution. Plugs are then dispersed in water andincubated at 37° C. overnight. Hemoglobin (Hb) levels in the plugs aredetermined using Drabkin's solution (e.g., from Sigma) according to themanufacturers' instructions. The amount of Hb in the plug is an indirectmeasure of angiogenesis as it reflects the amount of blood in thesample.

In addition, or alternatively, animals may be injected prior tosacrifice with a 0.1 ml buffer (preferably PBS) containing a highmolecular weight dextran to which is conjugated a fluorophore. Theamount of fluorescence in the dispersed plug, determinedfluorimetrically, also serves as a measure of angiogenesis in the plug.Staining with mAb anti-CD31 (CD31 is “platelet-endothelial cell adhesionmolecule”, “PECAM”) may also be used to confirm neovessel formation andmicrovessel density in the plugs.

In Vivo Assessment of Angiogenesis Inhibition and Anti-Tumor EffectsUsing the Matrigel® Plug Assay with Tumor Cells

In this assay, tumor cells, for example 1-5×10⁶ cells of the 3LL Lewislung carcinoma or the rat prostate cell line MatLyLu, are mixed withMatrigel® and then injected into the flank of a mouse following theprotocol described above. A mass of tumor cells and a powerfulangiogenic response can be observed in the plugs after about 5 to 7days. The anti-tumor and anti-angiogenic action of a compound in anactual tumor environment can be evaluated by including it in the plug.Measurement is then made of tumor weight, Hb levels or fluorescencelevels (of a dextran-fluorophore conjugate injected prior to sacrifice).To measure Hb or fluorescence, the plugs are first homogenized with atissue homogenizer.

Xenograft Models of Subcutaneous Tumor Growth

Human Ovarian Carcinoma

A2780 human ovarian cancer line was established from tumor tissue froman untreated patient. The A2780 cells are maintained as a monolayer inRPMI 1640 medium supplemented with 2 mM glutamine, 0.01 mg/mL bovineinsulin, and 10% FBS. (Hamilton, T C et al., Sem. Oncol. 1984;11:285-293; Behrens, B C et al., Cancer Res. 1987; 47:414-418). Twomillion A2780 are inoculated in the right flank of nude Balb/c femalemice. The A2780 tumor is staged to 50 to 200 mm³ range before treatmentis. The IgG control Ab as well as the anti-D2D3 uPAR mAbs areadministered by the intraperitoneal route at 10 mg/kg twice weekly onMonday and Friday. The cisplatin treatment group was staged to 1000 mm³;animals received 6 mg/kg once a week. Tumor volumes were measured twicea week. At the time of sacrifice, plasma is obtained and the tumorexcised from each animal. Half of the tumor is snap frozen forbiochemical assessment and the rest is placed in Zinc fixative forhistological assessment.

Human Lung Carcinoma

A549, human lung carcinoma (ATCC Catalog No. CCL-185) cell line, wasestablished through explant culture of lung carcinomatous tissue from a58-year-old Caucasian male (Giard, D J et al., J. Natl. Cancer Inst.51:1417-23 (1973)). A549 cells are maintained in Ham's F12K mediumsupplemented with 2 mM L-glutamine, 0.15% NaHCO₃, and 10% FBS.

About 10⁶ A549 carcinoma cells are inoculated in the right flank ofC.B-17/Sys (scid/scid) Severe Combined Immunodeficient (SCID) femalemice. Treatment is preferably initiated the day after tumor inoculation.The IgG control Ab (and the PBS control) as well as the anti-D2D3 uPARmAb ATN-658 are administered intraperitoneally 10 mg/kg twice weekly onMonday and Friday. Initially tumor volumes are measured once a week.When the volume in any treatment group exceeds 300 mm³, measurements areobtained twice a week.

At the time of sacrifice, plasma is obtained and the tumor excised fromeach animal. Half of the tumor is snap frozen for biochemical assessmentand the rest is placed in Zinc fixative for histological assessment.

Xenograft Model of Metastasis

The Abs and/or conjugates are tested for inhibition of late metastasisusing an experimental metastasis model such as that of Crowley et al.,Proc. Natl. Acad. Sci. USA 1993, 90 5021-5025). Late metastasis involvesthe steps wherein tumor cells attach and extravasate, invade locally,seed, proliferate and induce angiogenesis. Human prostatic carcinomacells (PC-3) transfected with a reporter gene, preferably the greenfluorescent protein (GFP) gene, but as an alternative with a geneencoding the enzymes chloramphenicol acetyl-transferase (CAT),luciferase or LacZ, are inoculated into nude mice. This approach permitsutilization of either of these markers (fluorescence detection of GFP orhistochemical calorimetric detection of the various enzymes) to followthe fate of these cells. Cells are injected, preferably iv, andmetastases identified after about 14 days, particularly in the lungs butalso in regional lymph nodes, femurs and brain. This mimics the organtropism of naturally occurring metastases in human prostate cancer. Forexample, GFP-expressing PC-3 cells (10⁶ cells per mouse) are injected ivinto the tail veins of nude (nu/nu) mice. Animals are treated with atest composition at 100 μg/animal/day given q.d. IP. Single metastaticcells and foci are visualized and quantitated by fluorescence microscopyor light microscopic histochemistry or by grinding the tissue andquantitative calorimetric assay of the detectable label.

Pharmaceutical and Therapeutic Compositions and their Administration

The compounds that may be employed in the pharmaceutical compositions ofthe invention include all of the polypeptide molecules, preferably Abs,described above, as well as the pharmaceutically acceptable salts ofthese compounds. Pharmaceutically acceptable acid addition salts of thecompounds of the invention containing a basic group are formed whereappropriate with strong or moderately strong, non-toxic, organic orinorganic acids by methods known to the art. Exemplary of the acidaddition salts that are included in this invention are maleate,fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate,benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide,sulfate, phosphate and nitrate salts.

Pharmaceutically acceptable base addition salts of compounds of theinvention containing an acidic group are prepared by known methods fromorganic and inorganic bases and include, for example, nontoxic alkalimetal and alkaline earth bases, such as calcium, sodium, potassium andammonium hydroxide; and nontoxic organic bases such as triethylamine,butylamine, piperazine, and tri(hydroxymethyl)methylamine.

As stated above, the compounds of the invention possess the ability toinhibit EC proliferation, motility, or invasiveness and angiogenesis,properties that are exploited in the treatment of cancer, in particularmetastatic cancer. A composition of this invention may be active per se,or may act as a “pro-drug” that is converted in vivo to the active form.

Therapeutically Labeled Compositions

In a preferred embodiment, the mAbs describe herein are “therapeuticallyconjugated” or “therapeutically labeled” (terms which are intended to beinterchangeable) and used to deliver a therapeutic agent to the site towhich the compounds home and bind, such as sites of tumor metastasis orfoci of infection/inflammation, restenosis or fibrosis. The term“therapeutically conjugated” means that the modified mAb is conjugatedto another therapeutic agent that is directed either to the underlyingcause or to a “component” of tumor invasion, angiogenesis, inflammationor other pathology. A therapeutically labeled polypeptide carries asuitable therapeutic “label” also referred to herein as a “therapeuticmoiety.” A therapeutic moiety is an atom, a molecule, a compound or anychemical component added to the peptide that renders it active intreating a target disease or condition, primarily one a associated withundesired angiogenesis. The therapeutic moiety may be bound directly orindirectly to the mAb. The therapeutically labeled mAb is administeredas pharmaceutical composition which comprises a pharmaceuticallyacceptable carrier or excipient, and is preferably in a form suitablefor injection.

Examples of useful therapeutic radioisotopes (ordered by atomic number)include ⁴⁷Sc, ⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At,²¹²Pb and ²¹⁷Bi. These atoms can be conjugated to the peptide directly,indirectly as part of a chelate, or, in the case of iodine, indirectlyas part of an iodinated Bolton-Hunter group. The radioiodine can beintroduced either before or after this group is coupled to the peptidecompound.

Preferred doses of the radionuclide conjugates are a function of thespecific radioactivity to be delivered to the target site which varieswith tumor type, tumor location and vascularization, kinetics andbiodistribution of the peptide carrier, energy of radioactive emissionby the nuclide, etc. Those skilled in the art of radiotherapy canreadily adjust the dose of the peptide in conjunction with the dose ofthe particular nuclide to effect the desired therapeutic benefit withoutundue experimentation.

Another therapeutic approach included here is the use of boron neutroncapture therapy, where a boronated peptide is delivered to a desiredtarget site, such as a tumor, most preferably an intracranial tumor(Barth, R F, Cancer Invest. 14:534-550 (1996); Mishima, Y (ed.), CancerNeutron Capture Therapy, New York: Plenum Publishing Corp., 1996;Soloway, A H et al., (eds), J. Neuro-Oncol. 33.1-188 (1997). The stableisotope ¹⁰B is irradiated with low energy (<0.025 eV) thermal neutrons,and the resulting nuclear capture yields α-particles and ⁷Li nucleiwhich have high linear energy transfer and respective path lengths ofabout 9 and 5 μm. This method is predicated on ¹⁰B accumulation in thetumor with lower levels in blood, endothelial cells and normal tissue(e.g., brain). Such delivery has been accomplished using epidermalgrowth factor (Yang. W et al., Cancer Res 57:4333-4339 (1997).

Other therapeutic agents which can be coupled to the mAbs according tothe method of the invention are drugs, prodrugs, enzymes for activatingpro-drugs, photosensitizing agents, nucleic acid therapeutics, antisensevectors, viral vectors, lectins and other toxins.

Lectins are proteins, commonly derived from plants, that bind tocarbohydrates. Among other activities, some lectins are toxic. Some ofthe most cytotoxic substances known are protein toxins of bacterial andplant origin (Frankel, A E et al., Ann. Rev. Med. 37:125-142 (1986)).These molecules binding the cell surface and inhibit cellular proteinsynthesis. The most commonly used plant toxins are ricin and abrin; themost commonly used bacterial toxins are diphtheria toxin and Pseudomonasexotoxin A. In ricin and abrin, the binding and toxic functions arecontained in two separate protein subunits, the A and B chains. Thericin B chain binds to the cell surface carbohydrates and promotes theuptake of the A chain into the cell. Once inside the cell, the ricin Achain inhibits protein synthesis by inactivating the 60S subunit of theeukaryotic ribosome Endo, Y. et al., J. Biol. Chem. 262: 5908-5912(1987)). Other plant derived toxins, which are single chain ribosomalinhibitory proteins, include pokeweed antiviral protein, wheat germprotein, gelonin, dianthins, momorcharins, trichosanthin, and manyothers (Strip, F. et al., FEBS Lett. 195:1-8 (1986)). Diphtheria toxinand Pseudomonas exotoxin A are also single chain proteins, and theirbinding and toxicity functions reside in separate domains of the sameprotein Pseudomonas exotoxin A has the same catalytic activity asdiphtheria toxin. Ricin has been used therapeutically by binding itstoxic α-chain, to targeting molecules such as Abs to enablesite-specific delivery of the toxic effect. Bacterial toxins have alsobeen used as anti-tumor conjugates. As intended herein, a toxic peptidechain or domain is conjugated to a compound of this invention anddelivered in a site-specific manner to a target site where the toxicactivity is desired, such as a metastatic focus. Conjugation of toxinsto protein such as Abs or other ligands are known in the art (Olsnes, S.et al., Immunol. Today 10:291-295 (1989); Vitetta, E S et al., Ann. Rev.Immunol. 3:197-212 (1985)).

Cytotoxic drugs that interfere with critical cellular processesincluding DNA, RNA, and protein synthesis, have been conjugated to Absand subsequently used for in vivo therapy. Such drugs, including, butnot limited to, daunorubicin, doxorubicin, methotrexate, and Mitomycin Care also coupled to the compounds of this invention and usedtherapeutically in this form.

The compounds of the invention, as well as the pharmaceuticallyacceptable salts thereof, may be incorporated into convenient dosageforms, such as capsules, impregnated wafers, tablets or injectablepreparations. Solid or liquid pharmaceutically acceptable carriers maybe employed.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline, water, dextrose, glycerol and the like. Similarly, the carrieror diluent may include any prolonged release material, such as glycerylmonostearate or glyceryl distearate, alone or with a wax. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, soft gelatin capsule, sterile injectable liquid (e.g., asolution), such as an ampoule, or an aqueous or nonaqueous liquidsuspension. A summary of such pharmaceutical compositions may be found,for example, in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton Pa. (Gennaro 18th ed. 1990).

The pharmaceutical preparations are made following conventionaltechniques of pharmaceutical chemistry involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral, parenteral, topical, transdermal,intravaginal, intrapenile, intranasal, intrabronchial, intracranial,intraocular, intraaural and rectal administration. The pharmaceuticalcompositions may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand so forth.

The present invention may be used in the diagnosis or treatment of anyof a number of animal genera and species, and are equally applicable inthe practice of human or veterinary medicine. Thus, the pharmaceuticalcompositions can be used to treat domestic and commercial animals,including birds and more preferably mammals, as well as humans.

The term “systemic administration” refers to administration of acomposition or agent such as the polypeptide, described herein, in amanner that results in the introduction of the composition into thesubject's circulatory system or otherwise permits its spread throughoutthe body, such as intravenous (i.v.) injection or infusion. “Regional”administration refers to administration into a specific, and somewhatmore limited, anatomical space, such as intraperitoneal, intrathecal,subdural, or to a specific organ. Examples include intravaginal,intrapenile, intranasal, intrabronchial (or lung instillation),intracranial, intra-aural or intraocular. The term “localadministration” refers to administration of a composition or drug into alimited, or circumscribed, anatomic space, such as intratumoralinjection into a tumor mass, subcutaneous (s.c.) injections,intramuscular (i.m.) injections. One of skill in the art wouldunderstand that local administration or regional administration oftenalso result in entry of a composition into the circulatory system, i.e.,so that s.c. or i.m. are also routes for systemic administration.Injectables or infusible preparations can be prepared in conventionalforms, either as solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection or infusion, or asemulsions. Though the preferred routes of administration are systemic,such as i.v., the pharmaceutical composition may be administeredtopically or transdermally, e.g., as an ointment, cream or gel; orally;rectally; e.g., as a suppository.

For topical application, the compound may be incorporated into topicallyapplied vehicles such as a salve or ointment. The carrier for the activeingredient may be either in sprayable or nonsprayable form.Non-sprayable forms can be semi-solid or solid forms comprising acarrier indigenous to topical application and having a dynamic viscositypreferably greater than that of water. Suitable formulations include,but are not limited to, solution, suspensions, emulsions, creams,ointments, powders, liniments, salves, and the like. If desired, thesemay be sterilized or mixed with auxiliary agents, e.g., preservatives,stabilizers, wetting agents, buffers, or salts for influencing osmoticpressure and the like. Preferred vehicles for non-sprayable topicalpreparations include ointment bases, e.g., polyethylene glycol-1000(PEG-1000); conventional creams such as HEB cream; gels; as well aspetroleum jelly and the like.

Also suitable for topic application as well as for lung instillation aresprayable aerosol preparations wherein the compound, preferably incombination with a solid or liquid inert carrier material, is packagedin a squeeze bottle or in admixture with a pressurized volatile,normally gaseous propellant. The aerosol preparations can containsolvents, buffers, surfactants, perfumes, and/or antioxidants inaddition to the compounds of the invention.

For the preferred topical applications, especially for humans, it ispreferred to administer an effective amount of the compound to anaffected area, e.g., skin surface, mucous membrane, eyes, etc. Thisamount will generally range from about 0.001 mg to about 1 g perapplication, depending upon the area to be treated, the severity of thesymptoms, and the nature of the topical vehicle employed.

Other pharmaceutically acceptable carriers for polypeptide compositionsof the present invention are liposomes, pharmaceutical compositions inwhich the active protein is contained either dispersed or variouslypresent in corpuscles consisting of aqueous concentric layers adherentto lipidic layers. The active polypeptide is preferably present in theaqueous layer and in the lipidic layer, inside or outside, or, in anyevent, in the non-homogeneous system generally known as a liposomicsuspension. The hydrophobic layer, or lipidic layer, generally, but notexclusively, comprises phospholipids such as lecithin and sphingomyelin,steroids such as cholesterol, more or less ionic surface activesubstances such as dicetylphosphate, stearylamine or phosphatidic acid,and/or other materials of a hydrophobic nature. Those skilled in the artwill appreciate other suitable embodiments of the present liposomalformulations.

Therapeutic compositions for treating tumors and cancer may comprise, inaddition to the peptide, one or more additional anti-tumor agents, suchas mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g.,cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim ortrimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosinearabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin;enzymes or enzyme inhibitors, e.g., asparaginase, topoisomeraseinhibitors such as etoposide; or biological response modifiers, e.g.,interferons or interleukins. In fact, pharmaceutical compositionscomprising any known cancer therapeutic in combination with the peptidesdisclosed herein are within the scope of this invention. Thepharmaceutical composition may also comprise one or more othermedicaments to treat additional symptoms for which the target patientsare at risk, for example, anti-infectives including antibacterial,anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.

The therapeutic dosage administered is an amount which istherapeutically effective, as is known to or readily ascertainable bythose skilled in the art. The dose is also dependent upon the age,health, and weight of the recipient, kind of concurrent treatment(s), ifany, the frequency of treatment, and the nature of the effect desired,such as, for example, anti-inflammatory effects or anti-bacterialeffect.

Therapeutic Methods

The methods of this invention may be used to inhibit tumor growth andinvasion in a subject or to suppress angiogenesis induced by tumors byinhibiting endothelial cell growth and migration. By inhibiting thegrowth or invasion of a tumor or angiogenesis, the methods result ininhibition of tumor metastasis. A vertebrate subject, preferably amammal, more preferably a human, is administered an amount of thecompound effective to inhibit tumor growth, invasion or angiogenesis.The compound or pharmaceutically acceptable salt thereof is preferablyadministered in the form of a pharmaceutical composition as describedabove.

Doses of the proteins (including Abs), peptides, peptide multimers,etc., preferably include pharmaceutical dosage units comprising aneffective amount of the peptide. Dosage unit form refers to physicallydiscrete units suited as unitary dosages for a mammalian subject; eachunit contains a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active material and the particular therapeuticeffect to be achieved, and (b) the limitations inherent in the art ofcompounding such an active compound for the treatment of, andsensitivity of, individual subjects

By an effective amount is meant an amount sufficient to achieve a steadystate concentration in vivo which results in a measurable reduction inany relevant parameter of disease and may include growth of primary ormetastatic tumor, any accepted index of inflammatory reactivity, or ameasurable prolongation of disease-free interval or of survival. Forexample, a reduction in tumor growth in 20% of patients is consideredefficacious (Frei III, E., The Cancer Journal 3:127-136 (1997)).However, an effect of this magnitude is not considered to be a minimalrequirement for the dose to be effective in accordance with thisinvention.

In one embodiment, an effective dose is preferably 10-fold and morepreferably 100-fold higher than the 50% effective dose (ED₅₀) of thecompound in an in vivo assay as described herein.

The amount of active compound to be administered depends on the precisepeptide or derivative selected, the disease or condition, the route ofadministration, the health and weight of the recipient, the existence ofother concurrent treatment, if any, the frequency of treatment, thenature of the effect desired, for example, inhibition of tumormetastasis, and the judgment of the skilled practitioner.

A preferred dose for treating a subject, preferably mammalian, morepreferably human, with a tumor is an amount of up to about 100milligrams of active polypeptide-based compound per kilogram of bodyweight. A typical single dosage of the peptide or peptidomimetic isbetween about 1 ng and about 100 mg/kg body weight. For topicaladministration, dosages in the range of about 0.01-20% concentration (byweight) of the compound, preferably 1-5%, are suggested. A total dailydosage in the range of about 0.1 milligrams to about 7 grams ispreferred for intravenous administration. The foregoing ranges are,however, suggestive, as the number of variables in an individualtreatment regime is large, and considerable excursions from thesepreferred values are expected.

An effective amount or dose of the peptide for inhibiting endothelialcell proliferation or migration in vitro is in the range of about 1picogram to about 5 nanograms per cell. Effective doses and optimal doseranges may be determined in vitro using the methods described herein.

The compounds of the invention may be characterized as producing aninhibitory effect on tumor cell or endothelial cell proliferation,migration, invasion, or on angiogenesis, on tumor metastasis or oninflammatory reactions. The compounds are especially useful in producingan anti-tumor effect in a mammalian host, preferably human, harboring atumor wherein angiogenesis inhibition results in reduction in size orgrowth rate of the tumor or destruction of the tumor. Preferably, thesubject is a human.

A longer example of a disease or condition against which the abovemethod is effective include primary growth of a solid tumor, leukemia orlymphoma; tumor invasion, metastasis or growth of tumor metastases;benign hyperplasia; atherosclerosis; myocardial angiogenesis;post-balloon angioplasty vascular restenosis; neointima formationfollowing vascular trauma; vascular graft restenosis; coronarycollateral formation; deep venous thrombosis; ischemic limbangiogenesis; telangiectasia; pyogenic granuloma; corneal disease;rubeosis; neovascular glaucoma; diabetic and other retinopathy;retrolental fibroplasia; diabetic neovascularization; maculardegeneration; endometriosis; arthritis; fibrosis associated with achronic inflammatory condition, traumatic spinal cord injury includingischemia, scarring or fibrosis; lung fibrosis, chemotherapy-inducedfibrosis; wound healing with scarring and fibrosis; peptic ulcers; abone fracture; keloids; or a disorder of vasculogenesis, hematopoiesis,ovulation, menstruation, pregnancy or placentation associated withpathogenic cell invasion or with angiogenesis.

A preferred disease or condition to be treated by the above method istumor growth, invasion or metastasis. This in includes brain tumors.Examples of such brain tumors are astrocytoma, anaplastic astrocytoma,glioblastoma, glioblastoma multiformae, pilocytic astrocytoma,pleiomorphic xanthoastrocytoma, subependymal giant cell astrocytoma,fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmicastrocytoma, oligodendroglioma, anaplastic oligodendroglioma,ependymoma, anaplastic ependymoma, myxopapillary ependymoma,subependymoma, mixed oligoastrocytoma and malignant oligoastrocytoma.

The method is also used to treat a uterine disease such as endometriosisand pathogenic ocular neovascularization such as that associated with,or a cause of, proliferative diabetic retinopathy, neovascularage-related macular degeneration, retinopathy of prematurity, sicklecell retinopathy or retinal vein occlusion.

Angiogenesis inhibitors may play a role in preventing inflammatoryangiogenesis and gliosis following traumatic spinal cord injury, therebypromoting the reestablishment of neuronal connectivity (Wamil, A W etal., Proc. Nat'l. Acad. Sci. USA 95:13188-13193 (1998)). Therefore, thecompositions of the present invention are administered as soon aspossible after traumatic spinal cord injury and for several days up toabout two weeks thereafter to inhibit the angiogenesis and gliosis thatwould sterically prevent reestablishment of neuronal connectivity. Thetreatment reduces the area of damage at the site of spinal cord injuryand facilitates regeneration of neuronal function and thereby preventsparalysis. The compounds of the invention are expected also to protectaxons from Wallerian degeneration, reverse aminobutyrate-mediateddepolarization (occurring in traumatized neurons), and improve recoveryof neuronal conductivity of isolated central nervous system cells andtissue in culture.

General Recombinant DNA Methods

General methods of molecular biology have been amply described in theart (Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd (orlater) Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,1989; Ausubel, F et al., Current Protocols in Molecular Biology, Vol. 2,Wiley-Interscience, New York, (current edition); Kriegler, Gene Transferand Expression: A Laboratory Manual (1990); Glover, D M, ed., DNACloning: A Practical Approach, vol. I & II, IRL Press, 1985; Alberts, B.et al., Molecular Biology of the Cell, 4th (or later) Ed., GarlandPublishing, Inc., New York, N.Y. (2002); Watson, J D et al., RecombinantDNA, 2nd Ed. (or later) Ed., WH Freeman & Co.; 2nd edition (1993); andOld, R W et al., Principles of Gene Manipulation: An Introduction toGenetic Engineering, 5th (or later) ed., Univ. of Calif. Press, Berkeley(1994).

Unless otherwise indicated, a particular nucleic acid sequence isintended to encompasses conservative substitution variants thereof(e.g., degenerate codon substitutions) and a complementary sequence. Theterm “nucleic acid” is synonymous with “polynucleotide” and is intendedto include a gene, a cDNA molecule, an mRNA molecule, as well as afragment of any of these such as an oligonucleotide, and further,equivalents thereof (explained more fully below). Sizes of nucleic acidsare stated either as kilobases (kb) or base pairs (bp). These areestimates derived from agarose or polyacrylamide gel electrophoresis(PAGE), from nucleic acid sequences which are determined by the user orpublished. Protein size is stated as molecular mass in kilodaltons (kDa)or as length (number of amino acid residues). Protein size is estimatedfrom PAGE, from sequencing, from presumptive amino acid sequences basedon the coding nucleic acid sequence or from published amino acidsequences.

Specifically, DNA molecules encoding the amino acid sequencecorresponding to the polypeptides of the present invention, or activevariants thereof, can be synthesized by the polymerase chain reaction(PCR) (see, for example, U.S. Pat. No. 4,683,202) using primers derivedthe sequence of the protein disclosed herein. These cDNA sequences canthen be assembled into a eukaryotic or prokaryotic expression vector andthe resulting vector can be used to direct the synthesis of the fusionpolypeptide or its fragment or derivative by appropriate host cells, forexample COS or CHO cells.

The term “nucleic acid” as used herein is intended to include suchfragments or equivalents. The nucleic acid sequences of this inventioncan be DNA or RNA.

Prokaryotic or eukaryotic host cells transformed or transfected toexpress the present polypeptides are within the scope of the invention.For example, the polypeptide may be expressed in bacterial cells such asE. coli, insect cells (baculovirus), yeast, or mammalian cells such asChinese hamster ovary cells (CHO) or human cells (which are preferredfor human therapeutic use of the transfected cells). Other suitable hostare known to those skilled in the art. Expression in eukaryotic cellsleads to partial or complete glycosylation and/or formation of relevantinter- or intra-chain disulfide bonds of the recombinant polypeptide.Examples of vectors for expression in yeast S. cerevisiae includepYepSec1 (Baldari et al., 1987, EMBO J. 6:229-234), pMFa (Kurjan et al.1982 Cell 30:933-943), pJRY88 (Schultz et al., 1987, Gene 54:113-123),and pYES2 (Invitrogen Corporation, San Diego, Calif.). Baculovirusvectors available for expression of proteins in cultured insect cells(SF 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell.Biol. 3:2156-2165) and the pVL series (Lucklow et al., (1989) Virology170:31-39). Generally, COS cells (Gluzman 1981 Cell 23:175-182) are usedin conjunction with such vectors as pCDM 8 (Aruffo et al., supra, fortransient amplification/expression in mammalian cells, while CHO(dhfr-negative CHO) cells are used with vectors such as pMT2PC (Kaufmanet al., 1987, EMBO J. 6:187-195) for stable amplification/expression inmammalian cells. The NS0 myeloma cell line (a glutamine synthetaseexpression system) is available from Celltech Ltd.

Construction of suitable vectors containing the desired coding andcontrol sequences employs standard ligation and restriction techniqueswhich are well understood in the art. Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and re-ligated inthe form desired. The DNA sequences which form the vectors are availablefrom a number of sources. Backbone vectors and control systems aregenerally found on available “host” vectors which are used for the bulkof the sequences in construction. For the pertinent coding sequence,initial construction may be, and usually is, a matter of retrieving theappropriate sequences from cDNA or genomic DNA libraries. However, oncethe sequence is disclosed it is possible to synthesize the entire genesequence in vitro starting from the individual nucleotide derivatives.The entire gene sequence for genes of length in the range of 500-1000 bpmay be prepared by synthesizing individual overlapping complementaryoligonucleotides and filling in single stranded nonoverlapping portionsusing DNA polymerase in the presence of the deoxyribonucleotidetriphosphates. This approach has been used successfully in theconstruction of several genes of known sequence. See, for example, Edge,Nature 1981, 292:756; Nambair et al., Science 1984, 223:1299; and Jay,J. Biol. Chem. 1984, 259:6311. Synthetic oligonucleotides are preparedby methods described in references cited above or by Beaucage et al.,Tetrahedron Lett. 1981, 22:1859; and Matteucci et al., J. Am. Chem. Soc.1981, 103:3185.

The components of the desired vectors can be excised and ligated usingstandard restriction and ligation procedures. Site-specific DNA cleavageis performed by treating with the suitable restriction enzyme (orenzymes) under conditions which are generally understood in the art, andthe particulars of which are specified by the manufacturer of thesecommercially available restriction enzymes. See, e.g., New EnglandBiolabs, Product Catalog. If desired, size separation of the cleavedfragments may be performed by standard polyacrylamide gel or agarose gelelectrophoresis techniques (e.g., Meth. Enzymol. (1980) 65:499-560).

Any of a number of methods are used to introduce mutations into thecoding sequence to generate variants if these are to be producedrecombinantly. These mutations include simple deletions or insertions,systematic deletions, insertions or substitutions of clusters of basesor substitutions of single bases. Modification of the DNA sequence bysite-directed mutagenesis is a well-known technique for which protocolsand reagents are commercially available (Zoller et al., Nucleic AcidsRes. 1982, 10:6487-6500; Adelman et al., DNA 1983, 2:183-193)). Theisolated DNA is analyzed by restriction and/or sequenced by the dideoxynucleotide method (Sanger, Proc. Natl. Acad. Sci. USA 1977, 74:5463;Messing, et al., Nucleic Acids Res. 1981, 9:3091 or Maxam et al., Meth.Enzymol., supra).

Vector DNA can be introduced into mammalian cells via conventionaltechniques such as calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming host cells can befound in Sambrook et al. supra and other standard texts. In fusionexpression vectors, a proteolytic cleavage site is introduced at thejunction of the reporter group and the target protein to enableseparation of the target protein from the reporter group subsequent topurification of the fusion protein. Proteolytic enzymes for suchcleavage and their recognition sequences include Factor Xa, thrombin andenterokinase.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example I Materials and Methods Cell Lines Expressing Proteins

The Drosophila expression system (DES™; Invitrogen, Inc.) utilizes theSchneider 2 (S2) cell line, derived from Drosophila melanogaster, andplasmid vectors for the expression of heterologous proteins. The plasmidvectors for expression in S2 cells are very versatile, allowinginducible expression of a protein driven by the metallothionein (MT)promoter. The same plasmid also allows the protein to be secreted fromthe cell into the surrounding media, greatly simplifying proteinpurification. Multiple copies of the vector can be stably inserted intothe genomic DNA of S2 cells, increasing levels of protein expression.Proteins expressed in S2 cells are minimally glycosylated, which isimportant for the generation of Abs directed against the proteincomponent of uPAR. Typical yields of protein following purification are25-50 mg/L with a purity of approximately 95 percent (FIG. 1). Celllines expressing the following proteins have been generated: suPAR, D1,D2D3, scuPA, ATF1-143, ATF1-135, Kringle47-143, and Kringle47-135. Inaddition, clones have been generated for suPAR in which the N-linkedglycosylation sites have been abolished.

Reagents

¹²⁵I was purchased as Na¹²⁵I (480-630 MBq [13-17 mCi] per μg iodine)from the Amersham Corp.

Tumor Cell Lines

The following cell and tumor lines were used: A549, HeLa, and A2780. TheA2780 human ovarian cancer line was established from tumor tissue froman untreated patient. A2780 cells are maintained as a monolayer in RPMI1640 medium supplemented with 2 mM glutamine, 0.01 mg/mL bovine insulin,and 10% FBS (supra). A549, human lung carcinoma, ATCC Catalog No.CCL-185, described above, are maintained in Ham's F12K mediumsupplemented with 2 mM L-glutamine, 0.15% NaHCO3, and 10% FBS.

A2780 cells (2×10⁶) were inoculated in the right flank of nude Balb/cfemale mice. The tumor was staged to 50 to 200 mm³ range beforetreatment was initiated. The IgG control Ab as well as the anti-D2D3uPAR mAbs were administered intraperitoneally at 10 mg/kg twice weeklyon Monday and Friday. The cisplatin treatment group was staged to 1000mm³; animals received 6 mg/kg once a week. Tumor volumes were measuredtwice a week.

A549 carcinoma cells (10⁶) were inoculated in the right flank ofC.B-17/Sys (scid/scid) female mice (scid: Severe CombinedImmunodeficient). Treatment was started the day after tumor inoculation.The IgG control Ab (and the PBS control) as well as the anti-D2D3 uPARmAb ATN-658 were administered intraperitoneally 10 mg/kg twice weekly onMonday and Friday. Initially tumor volumes were measured once a week.When the volume in any treatment group exceeded 300 mm³, measurementswere obtained twice a week.

At the time of sacrifice, plasma is obtained and the tumor excised fromeach animal. Half of the tumor is snap frozen for biochemical assessmentand the rest is placed in Zinc fixative for histological assessment.

Example II Anti-D2D3 mAbs

Immunization of Balb/c mice with the D2D3 domain of recombinant suPARconjugated to KLH generated a robust immune response. Subsequent fusionexperiments generated parental clones with specific cross-reactivitywith the D2D3 domain of uPAR as determined by western blotting and ELISAassays using recombinant proteins. These parental clones were subjectedto limiting dilution and a panel of mAbs specific for D2D3 was obtained.The properties of four of these Abs are summarized in Table 3. Isotypingidentified all clones as IgG1, κ. Specificity for uPAR was confirmed bywestern blotting. The affinity of the Abs was determined using directbinding assays. The majority of clones have affinities of 1 to 5 nM.

TABLE 3 Anti-D2D3 (uPAR) antibodies Western Blot Clone # Isotype (suPAR)K_(D) (nM) ATN-615 IgG1, κ + 2 ATN-658 IgG1, κ + 1 ATN-616 IgG1, κ + 5ATN-617 IgG1, κ + 3

The results of Western blotting experiments using two of these Abs,ATN-615 and ATN-658, are shown in FIG. 3. Both of the mAbs specificallyrecognize suPAR and, specifically, the D2D3 domain of uPAR.

The functional activity of the anti-D2D3 antibodies was tested inmigration assays. Previous experiments have demonstrated thatuPAR-expressing CHO cells migrate towards uPA in a modified Boydenchamber assay (FIG. 4) and that this migration is dependent of the GFDof uPA (not shown). As shown in FIG. 4, cell migration is inhibited by amAb specific for D2D3 as well as a rabbit polyclonal Ab directed againstuPAR. Interestingly, cell migration is also inhibited by anti-α5integrin Abs but not by anti-α6 integrin Abs. Taken together, these datasuggest that integrin α5β1 and uPAR are critical for uPA-inducedmigration.

The utility of the various anti-D2D3 Abs for diagnostic imaging ortargeting of therapeutic agents is dependent on their ability to binduPAR on the cell surface with high affinity. As shown in FIG. 5,iodinated Ab ATN-658 binds to HeLa cells with a K_(D) of approx. 1.5 nM.This is consistent with the K_(D) for this Ab determined in directbinding experiments (Table 3), indicating that binding is unaffected bythe labeling process.

uPA may be bound to the uPAR receptor on the surface of tumor orendothelial cells in vivo. Thus, Abs that bind to uPAR in the presenceof uPA therefore have additional utility as diagnostic or targetingagents. mAb ATN-658 does not inhibit the binding of scuPA to uPAR (FIG.6) on the surface of HeLa cells and is able to bind to HeLa cells in thepresence of scuPA. Thus, ATN-658 can target both occupied and unoccupiedreceptors on the cell surface.

The amino acid sequences of consensus V_(L) and V_(H) chains, includingthe three CDR regions of ANT-658 and ATN-615 were determined by standardmethods and have been set forth above, and therefore are not repeatedhere, although they should be considered as incorporated into thisexemplary disclosure.

Example III Binding of uPA to uPAR

Binding of uPA to uPAR was measured using ¹²⁵I-labeled uPA and HeLacells. HeLa cells express abundant amounts of uPAR but do not expressuPA. Briefly, 100 μg of scuPA was labeled with 100 μCi of [¹²⁵I]NaIusing IodoGen™ iodination reagent (Pierce Biotechnology Inc.).Unincorporated labeled NaI was removed from the labeled protein using asize exclusion column and the labeled protein eluted in Tris-bufferedsaline containing 0.1% bovine serum albumin (BSA). HeLa cells wereincubated with increasing concentrations of [¹²⁵I]-scuPA diluted in PBScontaining 0.1% BSA for 2 h at 4° C. Cells were washed extensively withPBS/0.1% BSA, the cell monolayers lysed with 1M NaOH and the totalnumber of bound counts determined. Specific binding was determined byincubating cells with [¹²⁵I]-scuPA in the presence of a large excess ofunlabeled scuPA. Binding was also performed with MDA-MB231 cells whichexpress both uPA and uPAR. To determine binding of scuPA, endogenous uPAis first removed from the surface of MDA-MB231 cells by washing with abuffer containing 0.1 M glycine/100 mM NaCl, pH 3 for 5 minutes at 4° C.This protocol was also used to determine binding of [¹²⁵I]-ATF to HeLacells. The ability of Abs to inhibit the binding of either [¹²⁵I]-scuPAor [¹²⁵I]-ATF binding to HeLa cells was determined by incubating cellswith increasing amounts of the unlabeled Ab for 15 minutes at 4° C.,prior to the addition of the [¹²⁵I]-labeled protein.

Example IV Inhibition of Cell Migration

Inhibition of cell migration by Abs specific for uPA or uPAR was testedusing a modified Boyden chamber assay as described previously (Tarui, Tet al., (2003) J. Biol. Chem., 278:29863-29872). Briefly, the lower sideof a Boyden chamber filter was coated with 500 nM uPA and serum freemigration buffer (Dulbecco's modified Eagle's medium containing 10 mMHepes and 0.5% bovine serum albumin) added to the lower chamber.uPAR-expressing CHO cells were resuspended in serum free migrationbuffer (8×10⁵ cells/ml) and 100 μl added to the upper chamber. To testthe ability of anti-uPA or anti-uPAR Abs to inhibit cell migration,cells were pre-incubated with 10 μg/ml Ab for 15 minutes prior toaddition to the upper chamber. The cells were then incubated at 37° C.in 5% CO₂ for 20 h. Cells migrating to the lower chamber were detectedby staining with 0.5% crystal violet and light microscopy.

Example V Assay for Antibodies that Recognize the Same Epitope asATN-658 Using Biotinylated ATN-658

The anti-D2D3 Ab, ATN-658, was biotinylated using EZ-link™sulfo-NHS-LC-biotin (Pierce Biotechnology Inc.) according to themanufactures instructions. Typically, a 20-fold molar excess of thebiotin-labeling reagent was used to label ATN-658 and unincorporatedbiotin removed from the labeled Ab using a size exclusion column. Toensure that the labeled Ab retained its affinity for uPAR,Biotin-ATN-658 was tested in an ELISA assay for binding to suPAR. BoundBiotin-ATN-658 was detected using HRP-conjugated streptavidin. Biotinlabeling did not reduce the affinity of ATN-658 for suPAR (FIG. 9). Toidentify Abs that recognize the same epitope as ATN-658 a competitionassay was established. Briefly, 96-well EIA/RIA high protein bindingplates were coated with 100 ng/well of suPAR overnight at 4° C. Afterthe blocking of non-specific binding with 1% casein, plates were washedwith PBS and Abs to be tested, diluted in PBS/0.1% casein containing 0.2nM Biotin-ATN-658, added to the appropriate wells. Plates were incubatedfor a further 1 h at room-temperature, washed extensively with PBS/0.05%Tween-20 and the bound Biotin-ATN-658 detected using HRP-conjugatedstreptavidin and the appropriate substrate (FIGS. 10A/10B).

Example VI Activities of mAbs In Vivo

Antibodies were tested for their ability to inhibit tumor growth in vivoin two models: the A549 non-small cell human lung cancer model and theA2780 ovarian cancer model using the protocols and conditions describedin Example I. Treatment was started the day after tumor inoculation. TheIgG control Ab (and the PBS control) as well as the anti-D2D3 UPAR mAbATN-658 was administered by the intraperitoneal route at 10 mg/kg twiceweekly on Monday and Friday.

ATN-658 significantly inhibited growth in both of these models (FIGS. 7and 8).

All the references cited above are incorporated herein by reference intheir entirety, whether specifically incorporated or not.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

In the event of any disagreement between the amino acid sequencesdisclosed above and those those in the electronic or paper SequenceListing attached hereto or filed later, the sequences above shall takeprecedence.

1. A polypeptide ligand that binds to a binary uPA-uPAR complex and to aternary complex of uPA-uPAR with an additional molecule X, which ligandis further characterized by the following properties: (a) does notsubstantially bind to (i) free uPA or (ii) the region of uPAR thatrecognizes and binds to uPA, so that the ligand does not inhibituPA-uPAR binding (b) does not substantially bind to any of thefollowing: (i) a uPA-X complex (ii) the uPA-recognizing and uPA-bindingregion of X, (iii) free uPA, and (iv) free X; and (c) does notsubstantially inhibit uPA binding with uPAR or with X. 2-6. (canceled)7. The ligand of claim 1 that is an antibody or antigen binding fragmentthereof.
 8. (canceled)
 9. The antibody of claim 7 that is a monoclonalantibody (mAb).
 10. (canceled)
 11. The antibody of claim 7, wherein theantibody or antigen-binding fragment comprises: (a) a V_(L) chaincomprising three CDR's which have the respective combination of aminoacids sequences (i) SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5; or (ii)SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; and (b) a V_(H) chaincomprising three CDR's which have the respective combination of aminoacids sequences (i) SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, or (ii)SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
 12. The antibody of claim7, wherein the antibody or antigen-binding fragment comprises: (a) aV_(L) chain has the sequence SEQ ID NO:1 or SEQ ID NO:9; and (b) a V_(H)chain has the sequence SEQ ID NO:2 or SEQ ID NO:10. 13-16. (canceled)17. The mAb of claim 9 that is selected from the group consisting of:(a) a mAb designated ATN-615 produced by hybridoma having ATCC Accession#PTA-8192); (b) a mAb designated ATN-658 produced by hybridoma havingATCC Accession #PTA-8191; (c) a mAb having essentially the sameantigen-binding characteristics as ATN-615; and (d) a mAb havingessentially the same antigen-binding characteristics as ATN-658.
 18. Theligand of claim 1 that, when bound to the uPA-uPAR complexes, inhibitsbinding of the complexes with another biological ligand for thesecomplexes. 19-23. (canceled)
 24. The ligand of claim 1 wherein thebinding of said ligand to the complexes interferes with and inhibits (a)uPAR mediated assembly of fibronectin, (b) binding of fibronectin or afragment thereof to an integrin or (c) the assembly of vitronectincomponents.
 25. The ligand of claim 1 that is (a) diagnosticallydetectably labeled or (b) labeled with, conjugated to, or fused to, atherapeutically active moiety, rendering said ligand therapeuticallyactive. 26-27. (canceled)
 28. The antibody ligand of claim 7 that is (a)diagnostically detectably labeled or (b) labeled with, conjugated to, orfused to, a therapeutically active moiety, rendering said ligandtherapeutically active.
 29. The mAb ligand of claim 9 that is (a)diagnostically detectably labeled or (b) labeled with, conjugated to, orfused to, a therapeutically active moiety, rendering said ligandtherapeutically active. 30-36. (canceled)
 37. A diagnostic compositioncomprising: (a) the labeled ligand of claim 25; and (b) a diagnosticallyacceptable carrier.
 38. The composition of claim 37 wherein the ligandis labeled with (a) a radionuclide selected from the group consisting of³H, ¹⁴C, ³⁵S, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁹Zr, ⁹⁷Ru, ⁹⁹Tc, ¹¹¹In, ¹²³I, ¹²⁵I,¹³¹I, ¹⁶⁹Yb and ²⁰¹Tl, (b) a PET-imagable agent, (c) an MRI-imagableagent, (d) a fluorescer or fluorogen, (f) a chromophore or chromogen,(g) a phosphorescer, (h) a chemiluminescer, or (i) a bioluminescer.39-40. (canceled)
 41. A therapeutic anti-angiogenic or anti-tumorpharmaceutical composition that inhibits undesired angiogenesis, tumorgrowth and/or tumor metastasis comprising: (a) an effective amount ofthe therapeutically active ligand of claim 25; and (b) apharmaceutically acceptable carrier.
 42. A therapeutic anti-angiogenicor anti-tumor pharmaceutical composition of that inhibits undesiredangiogenesis, tumor growth and/or tumor metastasis comprising: (a) aneffective amount of the therapeutically active antibody ligand of claim26; and (b) a pharmaceutically acceptable carrier.
 43. (canceled) 44.The therapeutic pharmaceutical composition of claim 41 wherein thetherapeutically active moiety is (a) a chemotherapeutic drug, (b) atoxin or other peptide or polypeptide fused to said ligand, or (c) atherapeutic radionuclide selected from the group consisting of ⁴⁷Sc,⁶⁷Cu, ⁹⁰Y, ¹⁰⁹Pd, ¹²⁵I, ¹³¹I, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²¹¹At, ²¹²Pb and²¹⁷Bi. 45-47. (canceled)
 48. A method for inhibiting cell migration,cell invasion, cell proliferation or angiogenesis, or for inducingapoptosis, comprising contacting cells associated with undesired cellmigration, invasion, proliferation or angiogenesis with an effectiveamount of the therapeutically active ligand of claim
 25. 49. A methodfor treating a subject having a disease, disorder or conditioncharacterized by undesired angiogenesis, tumor growth and/or tumormetastasis comprising administering to the subject an effective amountof a therapeutic pharmaceutical composition of claim
 41. 50. An assaymethod for detecting in a sample a substance suspected of having thebinding properties of the ligand of claim 1, comprising (a) contactingthe sample with uPA-uPAR complexes and determining binding of acomponent of said sample to the complexes; (b) contacting the samplewith free uPAR and determining binding of a component of said sample tothe uPAR. (c) comparing the binding of (a) and (b), wherein the presenceof binding in (a) and a substantial absence or significantly lowerbinding in (b) is indicative of the present of said substance in thesample. 51-52. (canceled)
 53. The method of claim 50 wherein thesuspected substance is an antibody or antigen binding fragment thereof.54-55. (canceled)
 56. An assay method for detecting in a sample asubstance suspected of having the binding properties of the ligand ofclaim 1, comprising (a) contacting the sample with uPA-uPAR-X complexesand determining binding of a component of said sample to the complexes;(b) contacting the sample with one or more of (i) uPA:X complexes (ii)uPA-uPAR complexes or (iii) uncomplexed X and determining binding of acomponent of said sample to uPA-X, uPA-uPAR or X; (c) comparing thebinding of (a) and (b), wherein the presence of binding in (a) and asubstantial absence or significantly lower binding in (b) is indicativeof the present of said substance in the sample. 57-58. (canceled)
 59. Anassay method for identifying an antibody or other ligand that binds tothe same epitope as does mAb ATN-615 or mAb ATN-658 comprising measuringthe ability of a sample suspected of containing said antibody or otherligand to competitively inhibit the binding of detectably labeledATN-615 or ATN-658 to (i) immobilized suPAR, (ii) immobilized suPAR D2D3domain, or (iii) an immobilized fragment of suPAR or D2D3 of suPARwherein at least about 20% competitive inhibition indicates that anantibody or ligand binds to said same epitope.
 60. A method foridentifying a peptide that is recognized by (a) mAbATN-615, (b) mAbATN-658, or (c) an antibody or other ligand that has the bindingspecificity of mAb ATN-615 or mAb ATN-658, which method comprisesmeasuring the ability of a sample suspected of containing said peptide,or a candidate peptide, to competitively inhibit the binding ofdetectably labeled mAb ATN-615 or mAb ATN-658 or said antibody or otherligand with the same binding specificity, to (i) immobilized suPAR, (ii)immobilized suPAR D2D3, or (iii) an immobilized fragment of suPAR orD2D3, wherein at least about 20% competitive inhibition indicates thatthe peptide has said binding specificity.
 61. An assay to screen for acompound that has, or to determine whether a candidate compound has,essentially the same binding characteristics when binding to a uPARstructure as does ATN-615 or ATN-658, comprising measuring the abilityof a sample being screened or the candidate compound to competitivelyinhibit the binding of detectably labeled ATN-615 or ATN-658 to (i)immobilized suPAR, (ii) immobilized suPAR D2D3, or (iii) an immobilizedfragment of suPAR or of D2D3, wherein, at least about 20% competitiveinhibition indicates that the compound has said binding characteristics.62-72. (canceled)